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balb / c female mice ( 68 week old ) were obtained from jackson laboratory . animal use conformed to institutional guidelines of weill cornell medical college and the feinstein institute for medical research . mice were immunized with multi - antigenic peptide coupled to dweysvwlsn ( mp , 100 g ) , control polylysine core ( mc , 100 g ) , scrambled wsdyevwlsn ( ms , 100 g ) , or aaaaavwlsn ( ma , 100 g , anaspec ) as previously described15 . we used mouse monoclonal aab r4a or control igg2b ( 200 g , southern biotech ) , human monoclonal aab g11 , human control aab b1 or human anti - dna ab d9 ( 150 g ) , obtained by isolating peptide - reactive or non - reactive b cells and expressing the ig heavy and light chain genes in 293 cells39,40 . the d form of dweysvwlsn peptide ( 200 g ) or irrelevant peptide nemqssrlre was given i.p . to mice at the time of intravenous r4a administration , and again 24 h and 48 h later . eu - labeled monoclonal abs ( perkin elmer ) were intravenously administered ( 100 g ) to gestating dams at e14 . fetal brains exposed to eu - r4a were stained with ab to cd-31 to identify blood vessels . for tunel assays , coronal sections ( 20 m thick ) of e15 brains were processed according to the manufacturer ( r&d systems ) and counterstained with methyl green . for ph3 assays of mitotic cells , frozen brain sections ( 20 m ) were blocked with 10% normal goat serum and incubated overnight at 4c with ph3 ab ( 1:1000 ; chemicon , millipore , billerica , ma ) followed by red x goat anti - rabbit igg ( jackson immuno research laboratories ) . measurements of the cp and the cerebral width were performed on nestin ( 1:200 ; chemicon ) and dapi ( 1:1000 ; invitrogen , carlsbad , ca ) stained e15 coronal sections , with openlab software ( improvision , perkinelmer ) . quantification of ph3 + and tunel+ cells was performed using automated image analysis programs on an axio - image microscope ( zeiss , thornwood , ny ) . anatomic regions were identified using within section coordinates and positive cells were counted in comparable areas of interest . negative geotaxis was measured23 in offspring born from mph ( n = 19 ) , mpl ( n = 14 ) and mc ( n = 15 ) dams at ages p11 , p15 or p16 and p20 . mice were placed with their heads facing downward on an incline ( 45 ) and the time to turn 180 and climb the incline was measured ( maximum 30 s , if the animal did not reach the top of the incline in the allotted time , a score of 30 s was assigned ) . each mouse was given three trials and the mean latency was calculated . adult male offspring from mph ( n = 10 ) , mpl ( n = 8) and mc ( n = 10 ) groups underwent a behavioral battery15,16 ( described in supplementary fig . we used a blinded format and randomized the order in which the cohorts of animals were tested in different tasks . the novel object recognition task27 consisted of three phases . in the first ( sample ) , mice explored two identical objects for 5 min . in the second ( delay ) , mice stayed in their home cage for 10 min . test objects replaced sample objects , so that one was identical to those in the sample phase ( familiar object ) and the other was different ( novel object ) . for the final phase ( choice ) , mice inspected the test objects for 5 min . object exploration was scored automatically with the pc - based anymaze software ( stoelting ) . we defined a3 as the exploration time for the familiar object , and b1 as the exploration time for the novel object , and measured the discrimination ratio , ( b1a3 ) / ( b1+a3 ) . the topological task28 consisted of five sessions ( 5 min ) with varying intersession intervals ( 3 min , 10 min , 3 min and 10 min ; sequentially ) . four different objects were arranged in a square . in sessions 1 and 2 , mice were familiarized to this arrangement . before session 3 , two objects were transposed , creating a novel configuration ii . animals were expected to explore displaced objects more avidly during session 3 and 4 . before session 5 , the two objects that had remained unchanged were switched , thus creating configuration iii . we defined d1 as the sum of the times exploring the displaced objects in configurations ii and iii ( sessions 3 and 5 ) and d2 as the sum of the times exploring the non - displaced objects ( sessions 3 and 5 ) . topological processing was measured with the ratio , ( d1d2 ) / ( d1+d2 ) . the fear extinction task26 initially required animals to associate the conditioned stimulus ( cs , 20-s tone , 5 khz , 80 db ) with the unconditioned stimulus ( us , foot - shock , 1 s , 0.6 ma ) , as previously described16 . extinction learning was tested 24 h later by exposing mice to 20 trials of cs alone in a novel chamber , with an intertrial interval of 120 s. to determine fear extinction , we measured the amount of freezing during each tone , using freezeframe software ( actimetrics ) . eight months after birth , mice that had been subjected to behavioral studies were anesthetized and perfused . the anterior striatum , and the anterior and posterior commissure were the internal landmarks to block and section the tissue in order to determine the volume of neocortical areas , hippocampus and amygdala . tissue was stained with cresyl violet and mouse anti - neun ( 1:1000 ; chemicon ) . internal cortical coordinates were the interhemispheric fissure and the anterior corpus callosum ; for hippocampus and amygdala ( basolateral and lateral nuclei ) , the coronal perimeters were captured on video screen by ibas software ( zeiss ) from the dorsal third ventricle to the posterior commissure . for neuron size determinations , video capture of neurons was performed using an optical dissector randomly sampling each region of interest . statistical comparisons were performed with kruskal - wallis and fisher 's analysis of variance , student 's t test ( unpaired , two - sided ) , mann - whitney u test , and kolmogorov - smirnov non - parametric test . we used the software packages spss ( spss inc . ) , statistica ( statsoft ) and origin ( originlab ) for our analyses . balb / c female mice ( 68 week old ) were obtained from jackson laboratory . animal use conformed to institutional guidelines of weill cornell medical college and the feinstein institute for medical research . mice were immunized with multi - antigenic peptide coupled to dweysvwlsn ( mp , 100 g ) , control polylysine core ( mc , 100 g ) , scrambled wsdyevwlsn ( ms , 100 g ) , or aaaaavwlsn ( ma , 100 g , anaspec ) as previously described15 . we used mouse monoclonal aab r4a or control igg2b ( 200 g , southern biotech ) , human monoclonal aab g11 , human control aab b1 or human anti - dna ab d9 ( 150 g ) , obtained by isolating peptide - reactive or non - reactive b cells and expressing the ig heavy and light chain genes in 293 cells39,40 . the d form of dweysvwlsn peptide ( 200 g ) or irrelevant peptide nemqssrlre was given i.p . to mice at the time of intravenous r4a administration , and again 24 h and 48 h later . eu - labeled monoclonal abs ( perkin elmer ) were intravenously administered ( 100 g ) to gestating dams at e14 . fetal brains exposed to eu - r4a were stained with ab to cd-31 to identify blood vessels . for tunel assays , coronal sections ( 20 m thick ) of e15 brains were processed according to the manufacturer ( r&d systems ) and counterstained with methyl green . for ph3 assays of mitotic cells , frozen brain sections ( 20 m ) were blocked with 10% normal goat serum and incubated overnight at 4c with ph3 ab ( 1:1000 ; chemicon , millipore , billerica , ma ) followed by measurements of the cp and the cerebral width were performed on nestin ( 1:200 ; chemicon ) and dapi ( 1:1000 ; invitrogen , carlsbad , ca ) stained e15 coronal sections , with openlab software ( improvision , perkinelmer ) . quantification of ph3 + and tunel+ cells was performed using automated image analysis programs on an axio - image microscope ( zeiss , thornwood , ny ) . anatomic regions were identified using within section coordinates and positive cells were counted in comparable areas of interest . negative geotaxis was measured23 in offspring born from mph ( n = 19 ) , mpl ( n = 14 ) and mc ( n = 15 ) dams at ages p11 , p15 or p16 and p20 . mice were placed with their heads facing downward on an incline ( 45 ) and the time to turn 180 and climb the incline was measured ( maximum 30 s , if the animal did not reach the top of the incline in the allotted time , a score of 30 s was assigned ) . each mouse was given three trials and the mean latency was calculated . adult male offspring from mph ( n = 10 ) , mpl ( n = 8) and mc ( n = 10 ) groups underwent a behavioral battery15,16 ( described in supplementary fig . we used a blinded format and randomized the order in which the cohorts of animals were tested in different tasks . the novel object recognition task27 consisted of three phases . in the first ( sample ) , mice explored two identical objects for 5 min . in the second ( delay ) , mice stayed in their home cage for 10 min . test objects replaced sample objects , so that one was identical to those in the sample phase ( familiar object ) and the other was different ( novel object ) . for the final phase ( choice ) , mice inspected the test objects for 5 min . object exploration was scored automatically with the pc - based anymaze software ( stoelting ) . we defined a3 as the exploration time for the familiar object , and b1 as the exploration time for the novel object , and measured the discrimination ratio , ( b1a3 ) / ( b1+a3 ) . the topological task28 consisted of five sessions ( 5 min ) with varying intersession intervals ( 3 min , 10 min , 3 min and 10 min ; sequentially ) . four different objects were arranged in a square . in sessions 1 and 2 , mice were familiarized to this arrangement . before session 3 , two objects were transposed , creating a novel configuration ii . animals were expected to explore displaced objects more avidly during session 3 and 4 . before session 5 , the two objects that had remained unchanged were switched , thus creating configuration iii . we defined d1 as the sum of the times exploring the displaced objects in configurations ii and iii ( sessions 3 and 5 ) and d2 as the sum of the times exploring the non - displaced objects ( sessions 3 and 5 ) . topological processing was measured with the ratio , ( d1d2 ) / ( d1+d2 ) . the fear extinction task26 initially required animals to associate the conditioned stimulus ( cs , 20-s tone , 5 khz , 80 db ) with the unconditioned stimulus ( us , foot - shock , 1 s , 0.6 ma ) , as previously described16 . extinction learning was tested 24 h later by exposing mice to 20 trials of cs alone in a novel chamber , with an intertrial interval of 120 s. to determine fear extinction , we measured the amount of freezing during each tone , using freezeframe software ( actimetrics ) . eight months after birth , mice that had been subjected to behavioral studies were anesthetized and perfused . the anterior striatum , and the anterior and posterior commissure were the internal landmarks to block and section the tissue in order to determine the volume of neocortical areas , hippocampus and amygdala . tissue was stained with cresyl violet and mouse anti - neun ( 1:1000 ; chemicon ) . internal cortical coordinates were the interhemispheric fissure and the anterior corpus callosum ; for hippocampus and amygdala ( basolateral and lateral nuclei ) , the coronal perimeters were captured on video screen by ibas software ( zeiss ) from the dorsal third ventricle to the posterior commissure . for neuron size determinations , video capture of neurons statistical comparisons were performed with kruskal - wallis and fisher 's analysis of variance , student 's t test ( unpaired , two - sided ) , mann - whitney u test , and kolmogorov - smirnov non - parametric test . we used the software packages spss ( spss inc . ) , statistica ( statsoft ) and origin ( originlab ) for our analyses . locally advanced breast cancer ( labc ) is defined by presence of a large primary tumor ( > 5 cm or t3 ) , associated with or without skin or chest - wall involvement ( t4 ) or with fixed ( matted ) axillary lymph nodes or with disease spread to ipsilateral internal mammary or supraclavicular nodes in the absence of any evidence of distant metastases . labc accounts for 10 - 20% in the west , while in india , it accounts for 30 - 35% of all cases . labc encompasses a wide spectrum of malignant breast tumors with varying presentation and poses a significant therapeutic challenge . the introduction of neoadjuvant chemotherapy ( nact ) in labc offered us advantages like initiation of early systemic therapy , delivery of drugs through intact vasculature , down - staging of tumors , which makes inoperable tumors operable and renders tumors suitable for breast conserving surgery ( bcs ) . it also helps in vivo assessment of response . national surgical adjuvant breast and bowel project ( nsabp)-18 and milan trials have shown that there were no difference in disease free survival ( dfs ) and overall survival between the patients who had received nact when compared to the patients who had received postoperative adjuvant chemotherapy . there are very few indian studies of nact in labc published until date . keeping this in mind , we have conducted a retrospective analysis to see the outcome of nact in labc patients at a tertiary care center . we retrospectively reviewed the case files of 148 patients of stage iii labc patients treated with nact followed by surgery and radiotherapy from january 2006 to december 2010 . the pathologic diagnosis was confirmed by fine - needle aspiration cytology or core needle biopsy performed before treatment . complete metastatic workup with chest x - ray , ultrasound abdomen , and bone scan of each patient was done . fac 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1.adriamycin - 50 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1 . ac t adriamycin - 60 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1paclitaxel - 175 mg / m iv day 1 cmf cyclophosphamide - 600 mg / m iv day 1.methotrexate - 40 mg / m iv day 1.5 fu - 600 mg / m iv day 1 . 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1.adriamycin - 50 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1 . 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1 . adriamycin - 60 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1paclitaxel - 175 mg / m iv day 1 adriamycin - 60 mg / m iv day 1 . cyclophosphamide - 600 mg / m iv day 1 paclitaxel - 175 mg / m iv day 1 cyclophosphamide - 600 mg / m iv day 1.methotrexate - 40 mg / m iv day 1.5 fu - 600 mg / m iv day 1 . clinical response ( cr ) to nact was assessed according to world health organization criteria . patients with positive or close margins were given boost with ir 192 implant or electron beam or conformal radiotherapy . postmastectomy radiation dose was 35 gy/15 # to chest - wall and 40 gy/15 # to the supraclavicular fossa . tamoxifen or letrozole was given to hormone receptor positive patients for 5 years according to the menopausal status . fac 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1.adriamycin - 50 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1 . ac t adriamycin - 60 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1paclitaxel - 175 mg / m iv day 1 cmf cyclophosphamide - 600 mg / m iv day 1.methotrexate - 40 mg / m iv day 1.5 fu - 600 mg / m iv day 1 . 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1.adriamycin - 50 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1 . 5 fluorouracil ( fu ) - 600 mg / m intravenous ( iv ) day 1 . adriamycin - 60 mg / m iv day 1.cyclophosphamide - 600 mg / m iv day 1paclitaxel - 175 mg / m iv day 1 adriamycin - 60 mg / m iv day 1 . cyclophosphamide - 600 mg / m iv day 1 paclitaxel - 175 mg / m iv day 1 cyclophosphamide - 600 mg / m iv day 1.methotrexate - 40 mg / m iv day 1.5 fu - 600 mg / m iv day 1 . cyclophosphamide - 600 mg / m iv day 1 . methotrexate - 40 mg / m iv day 1 . 5 fu - 600 mg / m iv day 1 . clinical response ( cr ) to nact was assessed according to world health organization criteria . patients with positive or close margins were given boost with ir 192 implant or electron beam or conformal radiotherapy . postmastectomy radiation dose was 35 gy/15 # to chest - wall and 40 gy/15 # to the supraclavicular fossa . tamoxifen or letrozole was given to hormone receptor positive patients for 5 years according to the menopausal status . median age at presentation was 46 years ( range : 22 - 72 years ) . 84 ( 56.7% ) patients presented with no axillary or single mobile ipsilateral axillary lymphnode , whereas 64 ( 43.30% ) patients had n2 or n3 disease . patient characteristics majority of the patients ( 60.8% ) patients received only anthracycline - based chemotherapy and 52 ( 35.1% ) patients received combination of anthracycline and taxane - based chemotherapy with median number of cycles being six . six patients received cmf based chemotherapy due to preexisting cardiac morbidity ( i.e. , coronary artery disease in three patients , dilated cardiomyopathy in one patient , systolic dysfunction with ejection fraction < 50% in two patients ) and of older age . these patients were of > 70 years of age and their mean karnofsky performance status ( ps ) was 70 . most of the patients ( 80.4% ) responded to nact either in the form of complete or partial response ( pr ) . complete cr was seen in18% patients and pr was seen in 62% patients , 13% patients had stable disease ( sd ) and 7% patients had progressive disease ( pd ) after nact . pathological complete response ( pcr ) was seen in 24 ( 16.20% ) patients [ table 2 ] . there was no significant difference in response when anthracycline and taxane - based chemotherapy was compared . most of the patients ( 78% ) underwent axillary dissection up to level ii . previously performing a mastectomy after nact was our institutional protocol . there was no statistically significant difference in dfs between the patients undergoing bcs and mastectomy . in 10 patients surgery median number of axillary lymphnodes dissected was 11 ( range : 3 - 19 ) . lymphovascular space invasion was positive in 43 ( 29.1% ) patients and extracapsular extension ( ece ) was present in 27 ( 18.2% ) patients . at a median follow - up period of 44 months , 36 patients ( 24.3% ) developed relapse of which six patients developed locoregional recurrence ( lrr ) while 28 patients developed distant metastases and two patients had recurrence in the contralateral breast . among six patients with lrr , three patients developed local recurrence , one patient developed axillary lymphnode recurrence , another two developed supraclavicular nodal relapse . patients with n0 or n1 disease had better 5 year dfs when compared to patients with n2 or n3 disease ( 84% vs. 48% ; p = 0.04 ) [ figure 1 ] . patients who had responded to chemotherapy ( cr + pr ) had significantly better 5 year dfs than nonresponders ( sd + pd ) ( 80% vs. 15% p = 0.02 ) . the patients who had achieved a pathological tumor size of <3 cm had better dfs when compared to patients with pathological tumor size of > 3 cm ( 93% vs. 22% p = 0.03 ) [ figure 2 ] . presence of ece was also associated with higher distant relapse . 5 year dfs was 24% in patients who had ece as compared to 89% in patient who had no ece . age , menopausal status , hormone receptor profile , lymphovascular space invasion , margin positivity had no significant impact on dfs as shown in table 3 . meier curve showing impact of nodal status on disease free survival kaplan meier curve showing impact of pathological tumour size on disease free survival kaplan meier curve showing impact of extracapsular extension on disease free survival impact of different prognostic factors median age at presentation was 46 years ( range : 22 - 72 years ) . 84 ( 56.7% ) patients presented with no axillary or single mobile ipsilateral axillary lymphnode , whereas 64 ( 43.30% ) patients had n2 or n3 disease . majority of the patients ( 60.8% ) patients received only anthracycline - based chemotherapy and 52 ( 35.1% ) patients received combination of anthracycline and taxane - based chemotherapy with median number of cycles being six . six patients received cmf based chemotherapy due to preexisting cardiac morbidity ( i.e. , coronary artery disease in three patients , dilated cardiomyopathy in one patient , systolic dysfunction with ejection fraction < 50% in two patients ) and of older age . these patients were of > 70 years of age and their mean karnofsky performance status ( ps ) was 70 . most of the patients ( 80.4% ) responded to nact either in the form of complete or partial response ( pr ) . complete cr was seen in18% patients and pr was seen in 62% patients , 13% patients had stable disease ( sd ) and 7% patients had progressive disease ( pd ) after nact . pathological complete response ( pcr ) was seen in 24 ( 16.20% ) patients [ table 2 ] . there was no significant difference in response when anthracycline and taxane - based chemotherapy was compared . breast conserving surgery was possible in 42 ( 28.4% ) cases . 96 ( 64.9% ) most of the patients ( 78% ) underwent axillary dissection up to level ii . previously performing a mastectomy after nact was our institutional protocol . there was no statistically significant difference in dfs between the patients undergoing bcs and mastectomy . in 10 patients surgery most ( 82.4% ) patients had invasive ductal carcinoma . only 12 ( 8.1% ) patients had positive margins . median number of axillary lymphnodes dissected was 11 ( range : 3 - 19 ) . lymphovascular space invasion was positive in 43 ( 29.1% ) patients and extracapsular extension ( ece ) was present in 27 ( 18.2% ) patients . at a median follow - up period of 44 months , 36 patients ( 24.3% ) developed relapse of which six patients developed locoregional recurrence ( lrr ) while 28 patients developed distant metastases and two patients had recurrence in the contralateral breast . among six patients with lrr , three patients developed local recurrence , patients with n0 or n1 disease had better 5 year dfs when compared to patients with n2 or n3 disease ( 84% vs. 48% ; p = 0.04 ) [ figure 1 ] . patients who had responded to chemotherapy ( cr + pr ) had significantly better 5 year dfs than nonresponders ( sd + pd ) ( 80% vs. 15% p = 0.02 ) . the patients who had achieved a pathological tumor size of <3 cm had better dfs when compared to patients with pathological tumor size of > 3 cm ( 93% vs. 22% p = 0.03 ) [ figure 2 ] . 5 year dfs was 24% in patients who had ece as compared to 89% in patient who had no ece . age , menopausal status , hormone receptor profile , lymphovascular space invasion , margin positivity had no significant impact on dfs as shown in table 3 . meier curve showing impact of nodal status on disease free survival kaplan meier curve showing impact of pathological tumour size on disease free survival kaplan meier curve showing impact of extracapsular extension on disease free survival impact of different prognostic factors there is paucity of literature pertaining to outcome of nact in labc in india where majority of breast cancer patients present with advanced disease . the aim of the study was to assess the use of nact in patients with labc at an indian tertiary care center . the median age of presentation in our study is 46 years , which is quite comparable with other studies . raina et al . in an early breast cancer study reported median age of 47 years whereas min et al . 42.6% of our patients were premenopausal , which is slightly lower than other studies by yadav et al . and a study by raina et al . showed estrogen receptor ( er ) positivity of 64% . there is a lot of variation in the number of cycles of chemotherapy that are given in neoadjuvant setting in the literature . investigators have administered either 3 - 4 cycles of chemotherapy or chemotherapy was continued up to maximal response . the advantage of giving chemotherapy up to maximal response is that if the patient has achieved good cr in less than planned cycles , continuation of further chemotherapy consolidates the complete response by maintaining the dose intensity . another advantage of continuing nact up to maximal response is that it may be possible that a fixed number of cycles may not be enough to achieve the amount of response necessary to do bcs and if chemotherapy is continued , further regression may continue . majority of our patients achieved maximal response after six cycles of nact . most of the patients ( 60.8% ) received anthracycline based chemotherapy as per institutional protocol . however , with increasing popularity of taxanes considerable amount of patients ( 35.1% ) received combined anthracycline and taxane - based chemotherapy . when docetaxel has been compared head on with anthracycline based chemotherapy it seems to show a better response rate in selected patients as reported in a small series . the nsabp trial has shown that use of taxanes with doxorubicin sequentially did show a better response rates in terms of superior partial and complete response both in er positive and negative patients . 80.4% of our patients responded to nact ( clinical cr + pr ) with pathological cr being 16.2% . our study showed a slightly lower pcr possibly because majority of patients received anthracycline based chemotherapy and we did not include patients with early breast cancer . the rates of bcs after nact in the present study is comparable to those reported in the literature . hence , bcs can be a good option after nact in labc patients instead of radical surgery . when we analyzed different prognostic factors , we found that response to chemotherapy was an important determinant of dfs . patients who responded to chemotherapy had significantly better dfs when compared with patients who had stable or pd after nact ( 80% vs. 15% ; p = 0.02 ) . deo et al . showed similar results in their study . in the present study , prognosis for patients with n0 or n1 disease was favorable compared with patients with advanced nodal disease ( 5 year dfs 84% vs. 48% ; p = 0.04 ) which was in concordance with the finding described by giordano . . showed pathological tumor stage did not have significant impact on lrr free survival . in a study by chen et al . it has been seen that 5 year lrr free survival was less in patients who had residual tumor size of > 2 cm after nact as compared to patients who had residual tumor size of < 2 cm . the current study showed patients who achieved pathological tumor size of <3 cm had better 5 year dfs than patients who had pathological tumor size of more than 3 cm ( 5 year dfs 93% vs. 22% ; p = 0.03 ) . brenner et al . showed borderline significance of ece in terms of survival , whereas yadav et al . we found presence of extensive ece was associated with decreased 5 year dfs ( 25% vs. 89% ; p = 0.025 ) . in the present study lrr was seen in 4% patients and ipsilateral breast tumor recurrence ( ibtr ) was seen only in three patients . the institut curie reported ibtr rates of 16% at 5 years and 22% at 10 years for patients who underwent bcs after nact . bonadonna et al . reported a 5 year ibtr rate of 7% after bcs and nact . in addition , cance et al . recently reported an ibtr rate of 10% among patients with advanced primary tumors treated with bcs after nact . most of our patients underwent modified radical mastectomy after nact which might explain very less ibtr rates in the present study . patient selection criteria , different therapeutic approaches , type of surgery , chemotherapeutic regimens used are probably the major factors responsible for the variation in published rate of lrr . the limitation of this study is its retrospective nature and its short follow - up . however bcs rates can be further increased with sequential use of taxanes in the future , which may also help us to get the real picture of locoregional relapse pattern after bcs in labc . the present study demonstrates clinicopathological variables such as nodal status , response to chemotherapy , pathological tumor size and presence of ece had significant impact on dfs .

systemic lupus erythematosus ( sle ) is an autoimmune disease mediated by autoantibodies ( aabs ) and preferentially affecting women of childbearing age . since the offspring of mothers with sle exhibit a high frequency of learning disorders1 - 5 , we hypothesized that maternally transferred aabs that bind dna and the n - methyl - d - aspartate receptor ( nmdar)6 - 12 could play a pathogenic role during fetal brain development . here we describe a maternal sle murine model wherein pregnant dams harbored dna - specific , nmdar - specific aabs throughout gestation . high titers of these aabs in maternal circulation led to histological abnormalities in fetal brain and subsequent cognitive impairments in adult offspring . these data support a paradigm in which in utero exposure to neurotoxic aabs causes abnormal brain development with long - term consequences . this paradigm may apply to multiple congenital neuropsychiatric disorders . background : introduction of neoadjuvant chemotherapy ( nact ) has dramatically changed the management of locally advanced breast cancer ( labc ) . however , very few randomized trials of nact have been carried out specifically in labc patients in our country . in this retrospective analysis , we presented our experience with nact in labc patients.materials and methods : medical records of 148 patients of stage iii labc patients treated with nact , followed by surgery and radiotherapy from january 2006 to december 2010 were reviewed . clinical and pathological responses to different chemotherapy regimens were assessed according to world health organization criteria . various factors influencing response to nact and clinical outcome were identified and analyzed.results:a total of 90 ( 60.8% ) patients received anthracycline - based chemotherapy and 52 ( 35.1% ) patients received mixed anthracycline and taxane - based chemotherapy.119 patients ( 80.4% ) responded to nact either in the form of complete or partial response ( pr ) . complete response was seen in 27 ( 18.2% ) patients and 92 ( 62.2% ) patients showed pr after nact . pathological complete response was seen in 24 ( 16.2% ) patients- . at a median follow - up period of 44 months 36 patients ( 24.3% ) developed relapse of which six patients developed locoregional recurrence , while 28 ( 18.9% ) patients developed distant metastasis . nodal status , response to chemotherapy , pathological tumor size <3 cm and extracapsular extension ( ece ) came out to be important prognostic factors in this study.conclusion:neoadjuvant chemotherapy is a reasonable alternative to upfront surgery in the management of labc . clinicopathological variables such as nodal status , response to chemotherapy , pathological tumor size and presence of ece had significant impact on disease free survival .

hearing loss is among the three most prevalent conditions worldwide , with around 636.5 million people suffering from a certain degree of hearing loss1 . it is estimated that in brazil , 6.8% of the population suffers from disabling hearing loss , i.e. , a hearing threshold level for the better ear of 41 db hl or greater , averaged at frequencies of 0.5 , 1 , 2 , and 4 khz . of these individuals , 5.3% are children between 4 and 9 years old , 36.2% are elderly , and 19.5% are adults between 20 and 59 years old2 . in the state of so paulo , a population - based study showed that the prevalence of hearing loss was 44% , being higher for elderly male individuals3 . a population - based cross - sectional study performed in rio de janeiro showed that , for the elderly , the prevalence of hearing loss in the better ear was 42.9%4 . hearing loss can hinder or even impede acquisition and development of oral language , as well as academic and social development in children . in addition , it can decrease the quality of life and work and educational opportunities of adult individuals . this condition also has an impact on society in terms of loss of productivity of affected individuals as well as costs of treatment , education , and rehabilitation of people with special needs . thus , healthcare actions focused on prevention , early identification , and treatment of hearing problems also have social and economic implications . the brazilian national health system ( sistema nico de sade , sus ) has incorporated the treatment of the hearing impaired in previous decades . such action was supplemented by the establishment of the national hearing healthcare policy in 20045 and , more recently , by the care network for people with disabilities within the sus6 . this network is organized into the components of primary care , specialized rehabilitation care , and emergency hospital care . primary care services are provided through healthcare basic units ( ubs ) , which prioritize certain strategic actions intended to enhance access for and attention to people with disabilities , including the promotion of early identification services . in brazil it is estimated that 910 out of 1000 school aged children have permanent hearing loss in at least one ear8 . the health in school program ( pse ) , initiated in 2007 as a joint action between the ministries of health and education , aims to incorporate the school community into projects and programs that make use of healthcare and education in order to comprehensively deal with the issues that compromise the development of brazilian children and teenagers . the primary task of the pse is the assessment of the health condition of students , including hearing status . in order to do so , audiometric procedures , among others , are recommended9 . for adults and the elderly , hearing loss onset is often insidious , complicating self - perception of the problem and , consequently , the request for treatment . performing hearing screening in this population would enable timely identification and corresponding referral to rehabilitation services , in order to minimize secondary consequences such as functional decline , depression , and social isolation . in brazil , there are still no large scale hearing screening programs targeting school aged children , adults , and the elderly . when screenings are conducted in this population , they are isolated events . the related costs can be attributed to the cost of the professional performing the procedure , the necessary time to perform it , and the cost of the necessary equipment . in order to decrease screening costs , a simple , fast , and effective method using low cost equipment that can be easily operated by a primary care professional it is also necessary to emphasize the difficulty of access to hearing health for populations living far from urban centers . thus , a relevant aspect of this scenario is the development of computer - based systems that can be used in telehealth models . different low cost systems for conducting audiometric screening11 12 13 or audiological assessment at distance ( 14 - 15 ) have been published in the literature . such systems use tdh-39 supra - aural headphones or er3a insert phones , which can increase device cost . one study was found on the use of a software - based screening audiometer that utilized a conventional circumaural phone with a 3.5 mm plug . however , this system was automatic and hearing responses were queried with 3 db increments , making the test relatively lengthy with a duration of about 15 minutes ( 10 ) . the telessade ( ts ) audiometer was developed to perform audiometric screening using off - the - shelf usb interface headphones . this characteristic has a relevant impact on acquisition and replacement costs ( the prototype costs less than 50 usd ) and also facilitates access of the user to the ts audiometer . a wide variety of usb headphones are available and the user can chose a model that is considered more convenient , once that set of headphones has been calibrated for use with the ts audiometer . the fact that only off - the - shelf electronic devices are used also facilitates the availability of the ts audiometer , as the eventual manufacturer will not require a specific infrastructure for manufacturing and distribution . the aim of this study was to evaluate the ts audiometer for use during audiometric screening . this randomized prospective study was conducted in the speech language pathology and audiology clinic of the x school , university x , and was approved by the relevant research ethics committee ( process number 021/2010 ) . once an informed consent form was signed by 60 individuals , aged 18 years or older , they were voluntarily enrolled in the study and segregated into 2 groups ( table 1 ) : group a : for this group , employees and graduate students of the bauru school of dentistry were invited to participate in the study . this group was composed of 30 adults with age varying from 18 to 41 years old . group b : for this group , individuals who had ent or audiological complaints and were referred to the clinic were invited to participate in the study . this group was comprised of 30 individuals with age ranging from 23 to 85 years old . there were 12 adults and 18 elderly subjects ( aged 60 years or older ) . the ts audiometer is integrated by software developed in visual basic.net 2005 ( net framework 2.0 ) for windows xp and a set of usb headphones . the system can store the registration information of the location where screening is performed , the information of the healthcare professionals ( users ) and screened populations , as well as the corresponding evaluation results . the audiometer uses a microsoft lifechat lx-3000 headset with the following specifications : ( a ) bilateral earphones ( 20 hz20 khz ) ; ( b ) embedded microphone ( 100 hz10 khz ) ; ( c ) embedded usb sound board with 16 bit precision ; ( d ) plug and play , i.e. , no other software installation is needed for normal use ; and ( e ) incorporated volume control . as for every usb device , the headset is identified by the host computer by its vendor i d and product i d , therefore allowing the software to apply the corresponding calibration parameter or impede the performance of the audiometric procedure if the corresponding parameters are not available . following fda recommendations for signature of electronic records 16 , the identity and electronic signature of the healthcare professional are protected by a password only known and modifiable by the healthcare professional . additionally , a change control , or audit trail , is not deemed necessary , as once they are electronically signed , the assessment results can no longer be modified . in this study , the ts audiometer was installed and tested in an asus eeepc900 laptop , given its relatively low cost and portability . this laptop has a 8.9 " screen , a celeron m353 processor , a 4 gb hard drive , 1 gb of ddr ii ram , an intel uma video board , a 1.3 megapixel webcam , a windows xp operating system , 802.11b / g wlan , usb / vga / earphone / mic / network inputs / outputs , embedded loudspeakers , a battery autonomy of approximately 2.5 hours , dimensions of 22.5 17 2 cm , and a weight of 0.99 kg . the ts audiometer includes a calibration user interface . these values are stored in the system parameters database and they are used every time an audiometric screening is performed . the device ( computer and headphones ) was calibrated by an engineer with experience in conventional audiometer calibration according to the applicable requirements of the standard project abnt / cb 03/ce-03:029.01 - 022/1 . the frequencies 2508000 hz were used to calibrate the left and right earphones of the microsoft lifechat lx-3000 headset . during calibration , the ts audiometer software was used to provide acoustic stimulation . with regard to the calibration , it must be noted that the usb headset included its own sound board , i.e. , its own analog and digital input / output stages . therefore , the calibration parameters were characteristic of the headset model and independent of the host computer model or type . the frequencies 250 , 500 , 1000 , 2000 , 3000 , 4000 , 6000 , and 8000 hz were calibrated ranging from 10 db hl ( minimum stimulation level ) to 70 db hl ( maximum stimulation level ) in 5 db steps for both the right and left channels . another feature contemplated during development of the ts audiometer was the real - time estimation of ambient noise levels during performance of the screening procedure . as audiometric screening is not necessarily conducted in an audiometric booth or an acoustically prepared room , the ts audiometer uses the microphone embedded in the headset , placed in the upright position , to determine the ambient noise level . the software application converts the sampled noise level into a db scale in order to assess if the real - time ambient noise level allows screening to be performed . the system will automatically display a message to the evaluator when the ambient noise level is greater than 60 db spl to make him / her aware that minimization of noise level is desirable . the system user interface will flag the responses obtained under an ambient noise level greater than 60 db spl and also stores the average ambient noise level measured during an evaluation on its database . this is intended to minimize the occurrence of false positives induced by the lack of an appropriate acoustic environment . as the screenings were conducted in a sound booth , audiometric screening was conducted in groups a and b with 2 different pieces of equipment : the sd 50 audiometer ( siemens ) coupled with supra - aural thd-39 headphones and the ts audiometer . the screenings were conducted on the same day by 2 different audiologists , each one operating one audiometer . the audiologists did not share the results obtained with each other and they were not aware of the patients ' hearing complaints . pure tones at frequencies of 500 , 1000 , 2000 , and 4000 hz were presented at a 25 db hl level . the participant was instructed to raise his / her hand each time the acoustic stimuli were heard . in the audiometric screening , fail when the subject did not respond to one or more stimuli presented in one or both ears . following audiometric screening , and regardless of the result ( pass or fail ) , all participants underwent otologic inspection , pure - tone threshold audiometry , and speech audiometry procedures . for such purposes , sd 50 ( siemens ) or midimate 622 ( madsen ) audiometers were used . air conduction hearing thresholds were obtained with tdh-39 earphones for the inter - octaves of the frequencies between 250 and 8000 hz , for both ears . when the air conduction hearing thresholds were greater than 20 db hl , bone conduction audiometry was also performed for the frequencies 500 , 1000 , 2000 , 3000 , and 4000 hz . hearing thresholds were determined by applying the ascendant - descendant strategy . at each pure - tone detection response , the presentation level was reduced by 10 db until the individual no longer responded to the stimuli . then , the presentation level was increased in 5 db steps until a response was detected . the hearing threshold , at each frequency , was the lowest level at which the individual could detect 50% of the stimuli presented . all procedures were performed in a sound booth , where the noise levels were within the specified ranges of the ansi 1999 standard 18 . concordance analysis and cohen 's kappa coefficient were used to analyze the screening results between the conventional and ts audiometers for groups a and b. the precision of the ts audiometer screening instrument was evaluated through specificity , sensitivity , positive predictive value , and negative predictive value analysis . the sensitivity was defined as the percentage of ears that failed screening with the ts audiometer among those in which hearing loss was observed with pure - tone threshold audiometry . the specificity was defined as the percentage of ears that passed screening among those with normal hearing results . in this study , air conduction audiometric thresholds less than or equal to 20 db hl were considered as normal hearing . the positive and negative predictive values were defined as the probability of a patient having hearing loss if they failed screening and the probability of a patient having normal hearing if they passing screening , respectively . with regard to group b , the number of individuals that failed the screening was 27 with the ts audiometer and 26 with the conventional audiometer . within group b , the other 26 participants presented different degrees of sensorineural hearing loss ( figure 2 ) , as per the pure - tone and speech audiometry results . mean and standard deviation of audiometric thresholds for participants who presented hearing loss ( n = 26 ) . in group a ( table 2 ) , all tested ears passed the screening . although the sample size was limited , this observation is supported by the fact that the participants were predominantly young females , a population for which the prevalence of hearing loss is smaller 2 3 . the concordance between the screening results obtained with the conventional and ts audiometers was excellent . in group it is worth noting that this group of participants had some kind of otologic complaint , with 18 of them reporting hearing difficulties . this group was also mostly composed of elderly subjects , for whom a greater prevalence of hearing loss is observed 2 3 4 . the number of failures at the frequency of 500 hz was less than that for the other frequencies , which contradicts results reported in the literature 10 . this can be explained by the fact that the screening was conducted in a sound booth , this being a limitation for the generalization of the data from the current study . in fact , hearing screening is generally conducted in a non - acoustically isolated room . consequently , ambient noise can exert a masking effect over emitted signals , which is more significant at low frequencies , thus affecting screening results at these frequencies . for this reason , several screening protocols exclude the 500 hz test , although this frequency is relevant for the assessment of the impact of middle ear condition on hearing sensitivity . in table 3 , the kappa coefficients show an excellent concordance between the evaluators for the screening results obtained with the conventional and ts audiometers at the frequencies of 500 hz to 3000 hz , and substantial concordance for the frequency of 4000 hz 19 . the inter - evaluator reliability allows verification of the degree of correspondence between the independent evaluations of 2 or more evaluators classifying the same phenomena . therefore , it is a relevant indicator of the quality of the screening procedure with the ts audiometer . previously , kappa coefficients of 0.790.93 were observed when evaluating the concordance between audiometric screenings conducted with a portable audiometer and a conventional audiometer20 . another study10 indicated a kappa coefficient of 0.20 between screening performed with a software - based audiometer and a conventional one . the fact that this result is lower than the one obtained in the current study could be related to the population characteristics ( school aged children ) and the impact of ambient noise at the frequency of 500 hz , since when this was excluded from the analysis , a kappa value of 0.62 was obtained . another important difference is that an automatic screening procedure was employed in the previous study , in contrast with the conventional procedure utilized in the current study . in table 4 , it can be observed that audiometric thresholds for group a were within normal values . audiometric thresholds greater than 25 db hl were found for at least one frequency in 65.4% of the ears tested in group b. for this group , 26 participants ( 86.6% ) presented sensorineural hearing loss , with a greater involvement of the high frequencies ( figure 2 ) . this high incidence of hearing loss is due to the fact that the participants of this group were recruited from a specialized hearing healthcare clinic . it is relevant to note that all of the subjects who presented hearing loss underwent a complete audiological assessment and received corresponding treatment , including fitting of hearing aids . therefore , the sensitivity and specificity of the screening procedures with the conventional and ts audiometers ( table 5 ) were calculated using the results obtained with pure - tone threshold audiometry as a reference . it was not possible to calculate the sensitivity of the procedures for group a due to the fact that none of the participants failed the screening or presented hearing loss with pure - tone audiometry . for group b , the sensitivity and specificity values of the ts audiometer were similar to those of the conventional audiometer ( table 5 ) . elevated sensitivity and specificity values avoid the occurrence of false negatives and false positives , respectively . however , it must be remembered that no procedure is entirely accurate , i.e. , no procedure has a sensitivity and specificity equal to 100% . the sensitivity and specificity values of the ts audiometer were similar to those found in the literature . the sensitivity and specificity of a portable screening audiometer were reported to be 91.898.5% and 88.096.3% , respectively20 . studies involving the audioscope device with pure - tone sweep ( 500 to 4000 hz ) for hearing screening demonstrated a sensitivity of 9497% and a specificity of 6980%11 . with regards to affordable audiometers , the ts audiometer presented higher sensitivity and specificity values than those found in the literature . sensitivity and specificity values of 86.7% and 75.9% were verified with a remote automatic audiometric screening method ( 20 db hl pure - tone sweep ) , based on the use of a computer and tdh-39 earphones 13 . automatic hearing screening conducted with a low cost audiometer and a circumaural phone showed a sensitivity of 100% and a specificity of 49% 10 . the differences observed between the literature and the present study can be related to the stimuli loudness level applied in the current study ( 25 db hl ) , attenuation differences in the earphones utilized , and , mainly , the screening method ( automatic vs. manual ) and screening room acoustics ( non - acoustically isolated room vs. audiometric booth ) used . under operational conditions ( field application ) , the performance indicators of a test procedure are modified by the frequency of occurrence of a medical condition within the population ( prevalence ) . therefore , the predictive value of the procedure has a significant relevance , i.e. , the probability of occurrence of a medical condition given a positive or negative result . in the current study , the positive and negative predictive values of the ts audiometer were equal to 94.9% and 91.5% , respectively , being similar to those obtained with a conventional audiometer ( table 5 ) . this means that if a subject fails screening with the ts audiometer , there is a 94.9% chance of them actually suffering from hearing loss and a 5.1% ( 100 - 94.9 ) chance of them having normal hearing . if the subject passes screening , there is 91.5% chance of them having normal hearing and an 8.5% ( 100 - 91.5 ) chance of them having hearing loss . for group b , the observed predictive values of the ts audiometer were greater than those found in the literature for other affordable audiometers , which presented positive and negative predictive values of 48.1% and 95.7%13 and 56% and 43%10 , respectively . these findings are justified by the differences observed in the sensitivity and specificity of the ts audiometer when compared to other audiometers , and the fact that the screened population was from a specialized hearing healthcare clinic , where a higher number of subjects with hearing loss were registered . screening programs are justified if there is evidence that sustains 3 standard criteria : ( a ) negative consequences of the medical condition must be sufficiently significant in order to justify the screening effort ; ( b ) an effective treatment for the detected medical condition is available ; and ( c ) an accurate , practical , and convenient screening test is available11 . although this study is limited by the number of participants , the results obtained suggest that the ts audiometer meets the necessary criteria for a screening procedure the specificity , sensitivity , and positive and negative predictive values of audiometric screening conducted with the ts audiometer were high and similar to those obtained with a conventional audiometer . this retrospective , hospital - based , observational study was carried out from august 2014 to july 2015 after obtaining necessary permission from the institutional ethics committee . diagnosed all patients admitted to the department of clinical haematology of scb medical college during the study period being treated with l - asp - based chemotherapy ( mcp 841 protocol ) all newly diagnosed indoor patients on induction therapy with the mcp 841 protocol , of either gender and up to 40 years of age , were included in the analysis . patients on any other drug other than those in the mcp 841 protocol that could have altered the blood glucose levels were excluded from the study . patients having abnormal plasma glucose level before starting therapy with l - asp were also excluded from the study . patients bed head tickets were collected from the record section of the hospital . under the mcp 841 protocol , a patient is on l - asp intramuscular every other day on days 220 of the first induction therapy . time of onset of hypo / hyperglycemia , persistence of plasma glucose alteration , and patient outcome were noted . fasting plasma glucose ( fpg ) levels were noted before the start of treatment with l - asp , on day 6 and day 12 of l - asp therapy and after the stoppage of l - asp therapy . differences in adr characteristics of native and pegylated l - asp were also noted and compared . the serum amylase levels of all these patients were estimated to exclude the possible pancreatitis . paired sample t - test was applied using medcalc version 12 - 1993 - 2013 medcalc software bvba ( acacialaan 22,8400 ostend , belgium ) to compare the fpg of patients before treatment with l - asp and on day 6 of l - asp treatment . paired sample t - test was applied using medcalc version 12 - 1993 - 2013 medcalc software bvba ( acacialaan 22,8400 ostend , belgium ) to compare the fpg of patients before treatment with l - asp and on day 6 of l - asp treatment . out of the 146 cases included for analysis , 129 were on native l - asp and only 17 were on pegylated l - asp . twenty - one percent of patients showed altered plasma glucose level out of 146 cases ( thirty in number ) . a total of 17 patients developed hypoglycemia ( fpg < 65 mg / dl ) whereas only 13 patients developed hyperglycemia ( fpg > 100 mg / dl ) . the serum amylase levels of all these patients were normal which showed the absence of pancreatitis . the median age in the hypoglycemia group was 5.5 years which is much less than that in the hyperglycemia group ( 19.5 years ) . the male : female ratio was 13:4 in hypoglycemia group and 10:3 in hyperglycemia group [ table 1 ] . demographic characteristics of patients on l - asparaginase of patients who were on native l - asp , 20.9% developed either hypo- or hyper - glycemia . on the contrary , in patients who were on pegylated l - asp all of them had hyperglycemia ( fpg > 100 mg / dl ) [ figure 1 ] . percentage of patients with plasma glucose alteration out of the total thirty patients who developed abnormality in fpg , 56.67% developed hypoglycemia whereas 43.33% had hyperglycemia . in majority of the cases , the fpg abnormality developed after taking three doses of l - asp in a particular cycle of chemotherapy . the number of patients who developed hypo- and hyper - glycemia after administration of native l - asp was 17 and 10 , respectively . on the contrary , all patients in hypoglycemic group had normal plasma glucose level before starting the treatment ( mean = 77.5 mg / dl ) . most of them developed hypoglycemia ( mean = 60.6 mg / dl ) on day 6 which returned to normal ( mean = 67.3 ) after stoppage of l - asp treatment . similarly , patients in hyperglycemia group had a mean plasma glucose of 86.5 mg / dl . on day 6 , majority of the patients hyperglycemia persisted even after the stoppage of l - asp treatment ( mean = 121.6 mg / dl ) . when the mean plasma glucose levels before the start of treatment with l - asp were compared with the values on day 6 by paired t - test , the p values for the hypo- and hyper - glycemia groups were found to be 0.0012 and 0.0309 , respectively . l - asp , an enzyme , has a wide application in many therapeutic protocols , including all . during all treatment , glucose levels are routinely monitored because many patients develop hyperglycemia , presumably because of glucocorticoids and l - asp . unexpectedly , many of our patients experienced repeated fasting hypoglycemia during induction and maintenance therapy . since altered fpg level can change the clinical outcome of patients , it was thought imperative to probe the exact course of hypo / hyperglycemia so that appropriate measures can be taken . most patients were given native l - asp and only a few were given pegylated l - asp , may be due to cost factor . approximately , one - fifth of the patients on l - asp therapy developed abnormal plasma glucose levels . most of the patients who developed hyperglycemia belonged to higher age group ( median age = 19.5 years ) compared to patients who have developed hypoglycemia ( median age = 5.5 years ) . this may be due to the fact that , in patients with higher age group , pancreatic reserve is less compared to younger age group . however , there is no significant difference between male to female ratio so far l - asp - induced abnormal plasma glucose levels ( hypo- or hyper - glycemia ) are concerned . in this study , as per the american diabetes association 2016 guidelines , fpg 100125 mg / dl is taken as impaired fasting glucose and > 126 is diabetic . hypoglycemia is taken as < 65 mg / dl because research in healthy adults shows that mental efficiency declines slightly but measurably as blood glucose falls below 65 mg / dl . fpg abnormality was observed in 20.9% of the cases in native l - asp group whereas only in 17.6% in pegylated l - asp group . we could find only cases of hyperglycemia in pegylated l - asp group whereas incidences of both hypoglycemia and hyperglycemia were observed in native l - asp - treated group . almost all the patients developed statistically significant abnormal glucose level after 3 dose of l - asp which may be due to the fact that the destruction of the endocrine pancreas is dose - dependent . however , though hypoglycemia returned to normal level after the end of the chemotherapy cycle , hyperglycemia persisted for longer duration almost till the end of observation in our study in most patients . given the time relation between l - asp administration and abnormal plasma glucose levels , we presume that our patients experienced l - asp - induced hypoglycemia . tanaka et al . in 2012 have reported that inappropriate insulin secretion , normal free fatty acids , and low ketone bodies during severe hypoglycemia indicate hyperinsulinism , suggesting that l - asp - induced hypoglycemia is a result of insulin hypersecretion . there were no other drugs that could have potentially induced hypoglycemia during the study period . glucocorticoids are known to induce hyperglycemia through insulin resistance and gluconeogenesis during induction therapy for childhood all . we suspect that parallel use of glucocorticoids may mask the hypoglycemic effect of l - asp . misgar et al . in 2014 have reported a case of 15-year - old boy who developed l - asp - induced hypoglycemia . use of l - asp has recently become even more common in the adult all regimens . we believe that in addition to the effect of l - asp on leukemic cells , it is important to pay more attention to its pharmacological features and physiological mechanisms . greater knowledge of these effects will enable clinicians to understand and develop appropriate strategies for l - asp use in chemotherapy regimens . hsu et al . in 2002 reported one case of diabetic ketoacidosis and persistent hyperglycemia as long - term complications of l - asp - induced pancreatitis . mondal et al . in 2010 reported two cases of diabetic ketoacidosis with l - asp therapy . quintanilla - flores et al . in 2014 reported one case of acute pancreatitis and diabetic ketoacidosis following l - asp / prednisone therapy in all . hence , to the best of our knowledge , there are only two reported cases of hypoglycemia induced by l - asp therapy by tanaka et al . of tokyo , japan , and misgar et al . of kashmir , however , in this retrospective study of ours , we have got 17 hypoglycemia as well as 13 hyperglycemia cases indicating the adverse effects of l - asp . our study reveals that a significant percentage of cases ( 21% ) have developed abnormal plasma glucose level among the study population . most of them developed hypoglycemia and they were in lower age group , whereas the higher age group patients developed hyperglycemia . in comparison to peg l - asp , native one was associated with more incidences of abnormal fpg . in majority of the cases , the fpg abnormality developed after taking three doses of l - asp in induction phase of mcp 841 protocol . identification of hypoglycemia as an adverse effect will enable clinicians to understand and develop appropriate strategies for l - asp use in chemotherapy regimens . in addition to the effect of l - asp on leukemic cells , it is important to pay more attention to its pharmacological features and physiological mechanisms .

summary introduction : the implementation of hearing screening programs can be facilitated by reducing operating costs , including the cost of equipment . the telessade ( ts ) audiometer is a low - cost , software - based , and easy - to - use piece of equipment for conducting audiometric screening . aim : to evaluate the ts audiometer for conducting audiometric screening . methods : a prospective randomized study was performed . sixty subjects , divided into those who did not have ( group a , n = 30 ) and those who had otologic complaints ( group b , n = 30 ) , underwent audiometric screening with conventional and ts audiometers in a randomized order . pure tones at 25 db hl were presented at frequencies of 500 , 1000 , 2000 , and 4000 hz . a fail result was considered when the individual failed to respond to at least one of the stimuli . pure - tone audiometry was also performed on all participants . the concordance of the results of screening with both audiometers was evaluated . the sensitivity , specificity , and positive and negative predictive values of screening with the ts audiometer were calculated . results : for group a , 100% of the ears tested passed the screening . for group b , pass results were obtained in 34.2% ( ts ) and 38.3% ( conventional ) of the ears tested . the agreement between procedures ( ts vs. conventional ) ranged from 93% to 98% . for group b , screening with the ts audiometer showed 95.5% sensitivity , 90.4% sensitivity , and positive and negative predictive values equal to 94.9% and 91.5% , respectively . conclusions : the results of the ts audiometer were similar to those obtained with the conventional audiometer , indicating that the ts audiometer can be used for audiometric screening . objectives : the objective of this study was to evaluate any abnormal change in plasma glucose levels in patients treated with l - asparaginase ( l - asp)-based chemotherapy regimen in patients of acute lymphoblastic leukemia ( all).materials and methods : this retrospective , hospital - based study was conducted in patients of all , admitted to the clinical haematology department of a tertiary care hospital of odisha from august 2014 to july 2015 . indoor records of 146 patients on multi - centered protocol-841 were evaluated for any alteration in plasma glucose level , time of onset of hypo / hyperglycemia , and persistence of plasma glucose alteration.results:twenty-one percent of patients showed abnormal plasma glucose level . most of these patients developed hypoglycemia and were of lower age group . most of these patients developed hypoglycemia and were of lower age group , whereas a majority of higher age group patients developed hyperglycemia . in majority of the cases , abnormal glucose developed after three doses of l - asp . hypoglycemia subsided whereas hyperglycemia persisted till the end of our observation period.conclusions:l-asp produces more incidences of hypoglycemia than hyperglycemia in a good number of all patients towards which clinicians should be more vigilant . however , hyperglycemia persists for a longer duration than hypoglycemia .

the latest several decades have witnessed the progress in the treatment of severe sepsis and septic shock , acute organ dysfunction and consequential multiple organ dysfunction , however , cardiovascular dysfunction due to severe infection is still a major contributor to sepsis related morbidity and mortality . cardiovascular dysfunction induced by severe sepsis and septic shock is characterized by signs of distributive shock and septic cardiomyopathy consisting of bi - ventricular myocardial contractility impairment and diastolic dysfunction . the characteristics of septic cardiomyopathy include left ventricular dilatation , depressed ejection fraction and recovery during 710 days . in severe sepsis and septic shock , myocardial depression is the manifestation of septic cardiomyopathy and may attribute to the overwhelming production of inflammatory cytokines , mitochondrial dysfunction , and decreased myofibrillar sensitivity to calcium . rivers et al . suggested in 2001 that early goal - directed therapy ( egdt ) was effective for severe sepsis management , however , several recent studies , including the process and arise trials in 2014 and the promise trial in 2015 indicated that egdt did not improve outcomes compared to usual care . international sepsis guidelines have been adopted worldwide , and it is widely accepted that the standard treatment for sepsis should concentrate on infection control and optimization of hemodynamic parameters by fluid resuscitation and vasopressor therapy including noradrenaline and vasopressin . in addition , using dobutamine to increase the cardiac index is recommended by international sepsis guidelines . however , several studies have demonstrated that the use of dobutamine to increase cardiac output did not improve microcirculation , peripheral perfusion , or the outcome of septic shock patients , and even increased the 90-day mortality rate . another inotropic agent is levosimendan , a ca sensitizer and inodilator , which has been used successfully in the management of acute heart failure . levosimendan not only has inotropic and vasodilator effects , but also has anti - inflammatory and anti - apoptotic effects . in addition , meta - analysis has shown that levosimendan reduced mortality in critically ill patients and chronic advanced heart failure patients . therefore , the aim of the present study was to compare the effects of levosimendan and dobutamine on biomarkers of myocardial injury and systemic hemodynamics in patients with septic shock in the icu . this pilot study was conducted in the medical - surgical icu at tongde hospital of zhejiang province in hangzhou , china between march 2014 and january 2016 . it was conducted in strict accordance with the protocol approved by the ethics committee of tongde hospital of zhejiang province(hangzhou , china ) . all participants were recruited by tongde hospital of zhejiang province and they all ( or their guardians ) signed informed consents prior to enrollment . patients with low cardiac output ( left ventricular ejective fraction ( lvef 45% ) were enrolled within the first 24 hours from the onset of septic shock after having established normovolemia ( cvp=12 to 15 mmhg ) and mean arterial pressure ( map ) of at least 65 mmhg using norepinephrine , if needed . red blood cells were transfused when hemoglobin concentrations decreased to below 7 g / dl to elevate systemic oxygen supply . inclusion criteria were : ( 1 ) all participants were diagnosed with septic shock and established normovolemia , and used norepinephrine to maintain map of at least 65 mmhg ; ( 2 ) all participants had lvef 45% after fluid resuscitation and vasopressor therapy . exclusion criteria included : ( 1 ) onset of septic shock > 24 hours ; ( 2 ) patients younger than 18 years of age ; ( 3 ) lvef 45% before fluid resuscitation ; ( 4 ) patients with pre - existing cardiomyopathy , valvular heart disease , or heart failure ; ( 5 ) present or suspected acute coronary syndrome within recent two weeks ; ( 6 ) pregnancy ; and ( 7 ) ventricular outflow tract obstruction . all patients received mechanical ventilation using a volume - controlled mode with a tidal volume of 6 to 8 ml / kg of predicted body weight , and were sedated with midazolam and fentanil . according to the international sepsis guidelines , for septic shock treatment we used norepinephrine to maintain a mean arterial pressure ( map ) of at least 65 mmhg despite quantitative fluid resuscitation ( the variation of cvp from 12 to 15 mmhg ) within the first 24 hours from the onset of septic shock . after these goals were achieved , cardiac ultrasound scans were used to measure lvef immediately . finally , 38 patients with low cardiac output ( lvef 45% ) were enrolled and randomized ( by the use of a table of random numbers ) into two groups : a group receiving 0.2 g / kg / minute ( without a loading bolus dose ) of levosimendan ( levosimendan group , n=19 ) , and a group receiving 5 g / kg / minute of dobutamine ( dobutamine group , n=19 ) . during the 24-hour drug intervention period , all patients also received fluid therapy and norepinephrine to maintain normovolemia and map of more than 65 mmhg . after this period , levosimendan and dobutamine were discontinued and the attending icu physicians decided whether dobutamine should be started based on the hemodynamic status of the patients . in addition , in order to avoid the interference of depurative extracorporeal circulation on biomarkers of myocardial injury , all patients did not receive continuous blood purification ( cbp ) before or within 48 hour after enrollment . the treating physicians and echocardiographers were not blinded to the experimental procedure or the echocardiographic and laboratory results during the study . transthoracic echocardiography was performed before and again after fluid resuscitation and vasopressor therapy , and 24 hours after inclusion . all echocardiograms were performed by an expert echocardiographer not involved in patient care , using a vivid e9 ultrasound scanner and acquiring lvef ( modified simpson s rule ) . all patients were monitored using a pulse - indicated continuous cardiac output ( picco ) system ( pulsion medical system , munich , germany ) . they were required to have both a left femoral artery catheter and a right central venous catheter . the correct placement of the catheter insertion was further confirmed by chest radiography . a 5-french thermistor - tipped catheter ( pv2013l16 , pulsion medical system , german ) was inserted into the femoral artery and a central venous catheter ( cs-277202-e , arrow , usa ) was placed into a central vein ( jugular or subclavian vein ) ; both were connected to the picco system . thermodilution parameters and pulse contour parameter were obtained with the picco monitor , based on triplicate injections of 15 ml of cold isotonic saline 0.9% ( < 8c ) via the central venous catheter , and were recorded as the average of the three measurements . the corresponding ventilator function and perfusion parameters were observed and kept constant during the 6-hour period preceding the measurements . blood gas samples , including lactate samples , were obtained from the arterial catheters via 3 ml heparinized syringes ( pl67bp ; bd diagnostics , plymouth , uk ) anaerobically and analyzed on blood gas bedside machines ( abl800 : radiometer , copenhagen , denmark ) . the changes in heart rate ( hr ) , cvp , map , stroke volume index ( svi ) , cardiac index ( ci ) , left ventricular stroke work index ( lvswi ) , systemic vascular resistance index ( svri ) , intrathoracic blood volume index ( itbvi ) , global end diastolic volume index ( gedi ) , extravascular lung water index ( evlwi ) , oxygen delivery index ( do2i ) , oxygen consumption index ( vo2i ) , lvef , lactate , positive end - expiratory pressure(peep ) , and norepinephrine dose were recorded at baseline and 24 hours after randomization . we collected 3 ml venous blood samples from the two groups of patients at baseline and 24 hours after randomization and sent samples immediately to department of biochemistry at tongde hospital of zhejiang province to measure plasma levels of heart - type fatty acid binding protein ( hfabp ) , troponin i ( tni ) , and brain natriuretic peptide ( bnp ) . pass software ( version 11 ; ncss , llc ) was used to calculate sample size . sample size was determined by two - sample t - test power analysis using preliminary data obtained in our laboratory with the following assumptions : of 0.05 ( two - tailed ) , power of 80% , differences in the mean of hfabp between patients in the levosimendan and the dobutamine groups of 3.4 ng / ml , and a standard deviation of 1.4 ng / ml . therefore , we calculated that a sample size of 17 would provide 80% power of detecting a difference at a 0.05 level of significance . the multivariate liner regression analysis was used to assess the effect of levosimendan on biomarkers of myocardial injury ( hfabp , tni , and bnp ) and heart function ( levf and ci ) after controlling for age , gender , apacheii scores , sofa scores , and baseline values in the septic shock patients . data were expressed as mean standard deviation of the mean ( sdm ) for quantitative variables , and as count and percentages for qualitative values . distributions of the discrete variables were compared between the two treatment groups with either the chi - square test or fisher exact tests . two sample t - test was used to compared between the two groups ; and paired t - test was used for continuous variables before and after treatment . chicago , il , usa ) was used for statistical analysis ; all tests were 2-tailed and p<0.05 was considered to be statistically significant . this pilot study was conducted in the medical - surgical icu at tongde hospital of zhejiang province in hangzhou , china between march 2014 and january 2016 . it was conducted in strict accordance with the protocol approved by the ethics committee of tongde hospital of zhejiang province(hangzhou , china ) . all participants were recruited by tongde hospital of zhejiang province and they all ( or their guardians ) signed informed consents prior to enrollment . patients with low cardiac output ( left ventricular ejective fraction ( lvef 45% ) were enrolled within the first 24 hours from the onset of septic shock after having established normovolemia ( cvp=12 to 15 mmhg ) and mean arterial pressure ( map ) of at least 65 mmhg using norepinephrine , if needed . red blood cells were transfused when hemoglobin concentrations decreased to below 7 g / dl to elevate systemic oxygen supply . inclusion criteria were : ( 1 ) all participants were diagnosed with septic shock and established normovolemia , and used norepinephrine to maintain map of at least 65 mmhg ; ( 2 ) all participants had lvef 45% after fluid resuscitation and vasopressor therapy . exclusion criteria included : ( 1 ) onset of septic shock > 24 hours ; ( 2 ) patients younger than 18 years of age ; ( 3 ) lvef 45% before fluid resuscitation ; ( 4 ) patients with pre - existing cardiomyopathy , valvular heart disease , or heart failure ; ( 5 ) present or suspected acute coronary syndrome within recent two weeks ; ( 6 ) pregnancy ; and ( 7 ) ventricular outflow tract obstruction . all patients received mechanical ventilation using a volume - controlled mode with a tidal volume of 6 to 8 ml / kg of predicted body weight , and were sedated with midazolam and fentanil . according to the international sepsis guidelines , for septic shock treatment we used norepinephrine to maintain a mean arterial pressure ( map ) of at least 65 mmhg despite quantitative fluid resuscitation ( the variation of cvp from 12 to 15 mmhg ) within the first 24 hours from the onset of septic shock . after these goals were achieved , cardiac ultrasound scans were used to measure lvef immediately . finally , 38 patients with low cardiac output ( lvef 45% ) were enrolled and randomized ( by the use of a table of random numbers ) into two groups : a group receiving 0.2 g / kg / minute ( without a loading bolus dose ) of levosimendan ( levosimendan group , n=19 ) , and a group receiving 5 g / kg / minute of dobutamine ( dobutamine group , n=19 ) . during the 24-hour drug intervention period , all patients also received fluid therapy and norepinephrine to maintain normovolemia and map of more than 65 mmhg . after this period , levosimendan and dobutamine were discontinued and the attending icu physicians decided whether dobutamine should be started based on the hemodynamic status of the patients . in addition , in order to avoid the interference of depurative extracorporeal circulation on biomarkers of myocardial injury , all patients did not receive continuous blood purification ( cbp ) before or within 48 hour after enrollment . the treating physicians and echocardiographers were not blinded to the experimental procedure or the echocardiographic and laboratory results during the study . transthoracic echocardiography was performed before and again after fluid resuscitation and vasopressor therapy , and 24 hours after inclusion . all echocardiograms were performed by an expert echocardiographer not involved in patient care , using a vivid e9 ultrasound scanner and acquiring lvef ( modified simpson s rule ) . all patients were monitored using a pulse - indicated continuous cardiac output ( picco ) system ( pulsion medical system , munich , germany ) . they were required to have both a left femoral artery catheter and a right central venous catheter . a 5-french thermistor - tipped catheter ( pv2013l16 , pulsion medical system , german ) was inserted into the femoral artery and a central venous catheter ( cs-277202-e , arrow , usa ) was placed into a central vein ( jugular or subclavian vein ) ; both were connected to the picco system . thermodilution parameters and pulse contour parameter were obtained with the picco monitor , based on triplicate injections of 15 ml of cold isotonic saline 0.9% ( < 8c ) via the central venous catheter , and were recorded as the average of the three measurements . the corresponding ventilator function and perfusion parameters were observed and kept constant during the 6-hour period preceding the measurements . blood gas samples , including lactate samples , were obtained from the arterial catheters via 3 ml heparinized syringes ( pl67bp ; bd diagnostics , plymouth , uk ) anaerobically and analyzed on blood gas bedside machines ( abl800 : radiometer , copenhagen , denmark ) . the changes in heart rate ( hr ) , cvp , map , stroke volume index ( svi ) , cardiac index ( ci ) , left ventricular stroke work index ( lvswi ) , systemic vascular resistance index ( svri ) , intrathoracic blood volume index ( itbvi ) , global end diastolic volume index ( gedi ) , extravascular lung water index ( evlwi ) , oxygen delivery index ( do2i ) , oxygen consumption index ( vo2i ) , lvef , lactate , positive end - expiratory pressure(peep ) , and norepinephrine dose were recorded at baseline and 24 hours after randomization . we collected 3 ml venous blood samples from the two groups of patients at baseline and 24 hours after randomization and sent samples immediately to department of biochemistry at tongde hospital of zhejiang province to measure plasma levels of heart - type fatty acid binding protein ( hfabp ) , troponin i ( tni ) , and brain natriuretic peptide ( bnp ) . pass software ( version 11 ; ncss , llc ) was used to calculate sample size . sample size was determined by two - sample t - test power analysis using preliminary data obtained in our laboratory with the following assumptions : of 0.05 ( two - tailed ) , power of 80% , differences in the mean of hfabp between patients in the levosimendan and the dobutamine groups of 3.4 ng / ml , and a standard deviation of 1.4 ng / ml . therefore , we calculated that a sample size of 17 would provide 80% power of detecting a difference at a 0.05 level of significance . the multivariate liner regression analysis was used to assess the effect of levosimendan on biomarkers of myocardial injury ( hfabp , tni , and bnp ) and heart function ( levf and ci ) after controlling for age , gender , apacheii scores , sofa scores , and baseline values in the septic shock patients . data were expressed as mean standard deviation of the mean ( sdm ) for quantitative variables , and as count and percentages for qualitative values . distributions of the discrete variables were compared between the two treatment groups with either the chi - square test or fisher exact tests . two sample t - test was used to compared between the two groups ; and paired t - test was used for continuous variables before and after treatment . chicago , il , usa ) was used for statistical analysis ; all tests were 2-tailed and p<0.05 was considered to be statistically significant . figure 1 shows a flow diagram that quantifies progress through the trial . of the 72 patients that did not meet the inclusion criteria , five patients declined to participate , 11 patients were excluded from the study for other reasons ( six because of an onset of septic shock > 24 hours , three because of lvef 45% before fluid resuscitation , and two due to acute coronary syndrome within the recent two weeks ) . age , gender , apache ii scores , sofa scores , and type of infection were balanced between the two groups at admission . there were no difference in the duration of mechanical ventilation , length of stay in icu and hospital , or 28-day mortality between the two groups ( all p>0.05 ) . as shown in table 2 , no significant differences were observed in hr , cvp , map , svri , itbvi , gedi , peep , and norepinephrine dose ( all p>0.05 ) . compared to dobutamine , levosimendan increased ci ( p=0.001 ) , svi ( p=0.030 ) , lvswi ( p=0.002 ) , and lvef ( p=0.018 ) , and decreased evlwi and lactate ( p=0.012 and p=0.022 , respectively ) after 24 hours of intervention . in addition , levosimendan improved do2 and vo2 of systemic tissues ( p=0.040 and p=0.031 , respectively ) . compared with the dobutamine group , concentrations of hfabp , tni , and bnp were significantly decreased at 24 hours after intervention in the levosimendan group ( p<0.001 , p=0.025 , and p<0.001 , respectively , see figures 24 . ) multivariate regression analysis showed that compared to the dobutamine group , after adjusting for the patients age , gender , apache ii , sofa , and baseline of hfabp , tni , bnp , lvef , and ci , the levosimendan group had significantly decreased levels of hfabp , tni , and bnp ( p<0.001 , p=0.03 and p<0.001 , respectively , tables 35 ) , and increased lvef and ci ( p=0.03 and p<0.001 , respectively , see tables 6 , 7 ) figure 1 shows a flow diagram that quantifies progress through the trial . of the 72 patients that did not meet the inclusion criteria , five patients declined to participate , 11 patients were excluded from the study for other reasons ( six because of an onset of septic shock > 24 hours , three because of lvef 45% before fluid resuscitation , and two due to acute coronary syndrome within the recent two weeks ) . age , gender , apache ii scores , sofa scores , and type of infection were balanced between the two groups at admission . there were no difference in the duration of mechanical ventilation , length of stay in icu and hospital , or 28-day mortality between the two groups ( all p>0.05 ) . as shown in table 2 , no significant differences were observed in hr , cvp , map , svri , itbvi , gedi , peep , and norepinephrine dose ( all p>0.05 ) . compared to dobutamine , levosimendan increased ci ( p=0.001 ) , svi ( p=0.030 ) , lvswi ( p=0.002 ) , and lvef ( p=0.018 ) , and decreased evlwi and lactate ( p=0.012 and p=0.022 , respectively ) after 24 hours of intervention . in addition , levosimendan improved do2 and vo2 of systemic tissues ( p=0.040 and p=0.031 , respectively ) . compared with the dobutamine group , concentrations of hfabp , tni , and bnp were significantly decreased at 24 hours after intervention in the levosimendan group ( p<0.001 , p=0.025 , and p<0.001 , respectively , see figures 24 . ) multivariate regression analysis showed that compared to the dobutamine group , after adjusting for the patients age , gender , apache ii , sofa , and baseline of hfabp , tni , bnp , lvef , and ci , the levosimendan group had significantly decreased levels of hfabp , tni , and bnp ( p<0.001 , p=0.03 and p<0.001 , respectively , tables 35 ) , and increased lvef and ci ( p=0.03 and p<0.001 , respectively , see tables 6 , 7 ) septic cardiomyopathy , a kind of cardiovascular dysfunction induced by severe sepsis and septic shock , is manifested by low cardiac output and is closely related to higher mortality in septic shock patients [ 1618 ] . inotropic agents including dobutamine and levosimendan may show benefits in the treatment of cardiovascular dysfunction induced by sepsis . the major findings of the present study were that levosimendan increased lvswi and lvef , decreased evlwi and lactate , and improved tissue perfusion , hemodynamics , and cardiac function . however , levosimendan did not reduce the days on mechanical ventilation , lengths of stay in icu and hospital , or 28-day mortality . elevated concentrations of tni and bnp are frequently observed in patients with severe sepsis and septic shock even in the absence of an acute coronary syndrome ( acs ) . demonstrated the relationship between elevated bnp level and severity of sepsis regardless of congestive heart failure ; their findings also supported the utility of bnp level as a marker for mortality in septic shock . studies have shown that tni was sensitive and superior to creatinine kinase - mb ( ck - mb ) for the detection of myocardial injury in septic shock and after coronary angioplasty with or without stenting . hence , bnp and tni have been identified as cardiac biomarkers and predictors of cardiac dysfunction and death in septic patients and successfully used in icus . however , it is difficult to distinguish septic patients with or without cardiac dysfunction related to septic shock via utilization of bnp alone . furthermore , tiruvoipati et al . suggested tni was related to lower ejection fraction and higher need for inotropic / vasopressor support , but did not independently predict mortality in critically ill patients with severe sepsis . hfabp is a more sensitive and specific biomarker of myocardial injury than tni and bnp , which make it superior to tni and bnp for the assessment of recurrent or persistent myocardial injury . zhang et al . found that serum hfabp was frequently elevated among patients with severe sepsis and was associated with sepsis - related myocardial dysfunction , and elevated hfabp independently predicted 28-day mortality in severe sepsis .therefore , hfabp , tni , and bnp were utilized as biomarkers of myocardial injury for septic shock patients in the present study . this study demonstrated that both dobutamine and levosimendan reduced the concentrations of biomarkers of myocardial injury in critically ill patients with septic shock . but hfabp , tni , and bnp were lower in the levosimendan group than in the dobutamine group , which suggested that levosimendan could play a more important role in attenuating septic myocardial injury . wu et al . conducted a study in patients with acute myocardial infarction ( ami ) who received emergency percutaneous coronary intervention ( pci ) and found that the tni concentration decreased much more in the levosimendan group than in the placebo group , suggesting that the myocardium was suffering less damage . in myocardium injury and stunning , the activation of the ca - dependent protease results in degradation of tni partially and selectively during reperfusion . because levosimendan has the ability to increase responsiveness to calcium while keeping the cytosolic ca concentration unchanged , levosimendan prevented the degradation of tni , which could prevent the contractile dysfunction of myocardium stunning and damage . studies found that bnp levels were elevated in severe sepsis and septic shock patients and elevated bnp were related to myocardial dysfunction , global tissue hypoxia , and mortality . several mechanisms probably account for the elevated bnp levels in sepsis , including neuro - hormonal activation , volume resuscitation , and sepsis - induced biventricular dilation , as well as stimulation of lipopolysaccharide or pro - inflammatory cytokines , acute lung injury , and acute respiratory distress syndrome induced sepsis . levosimendan was found to have significantly decreased bnp levels at day 1 and day 3 in patients with decompensated heart failure ( dhf ) and renal dysfunction . feola et al . demonstrated that bnp concentrations decreased more significantly in a levosimendan group than a furosemide group . kyrzopoulos et al . suggested that the decreasing effect of levosimendan treatment on bnp was associated with its decreasing effect on end - diastolic cardiac wall tension . immunomodulatory and anti - inflammatory properties of levosimendan might also contribute to its effect on decreased bnp . these probably mechanisms of decreased tni and bnp concentrations could explain the changes of tni and bnp in our study . hfabp , a stable low molecular weight protein found in the cytoplasm of myocardial cells , is a sensitive and specific biomarker of myocardial injury . it is frequently elevated in patients with severe sepsis and septic shock and is associated with sepsis - related myocardial dysfunction . the probably mechanisms leading to elevated hfabp concentrations in sepsis and septic shock cases may be related to sepsis - induced myocardial dysfunction and elevated levels of free fatty acids due to an increased catabolism of glycogen and lipids during multiple organ dysfunction syndrome ( mods ) associated inflammatory responses . in addition , decreased renal function also contributes to the elevation of hfabp . in this study , compared to dobutamine , levosimendan significantly decreased hfabp levels in patients with cardiomyopathy associated with severe sepsis and septic shock . to date first , levosimendan improves inotropic function due to binding to the ca saturated troponin c of myocardial thin filament and diastolic function because it does not promote calcium flux into the cell . the improvement in systolic and diastolic function and myocardial oxygen supply atttributed to levosimendan may decrease hfabp concentration via preventing myocardial dysfunction associated sepsis . second , the anti - inflammatory and anti - apoptotic effects enable levosimendan to reduce catabolism of glycogen and lipids via mediating inflammatory responses and decreasing the level of hfabp during sepsis and septic shock . finally , levosimendan decreases hfabp through reducing renal injury and improving renal function and because levosimendan leads to augmentation of renal perfusion , increases in renal blood flow and increases glomerular capillary surface area and glomerular filtration rate ( gfr ) . the present study demonstrated that levosimendan had no adverse effects on hemodynamics , on the contrary , it elevated lvef , svi , and ci and did not increase the dose of norepinephrine needed . in addition , decreased evlwi and lactate , and increased do2i and vo2i in patients with myocardial dysfunction suggests that levosimendan improved pulmonary vascular permeability and tissue perfusion via elevated cardiac output , and the driving pressure of blood flow entering into microcirculation . in our study , however , although there were no significant difference in mechanical ventilation time , icu length of stay , hospital length of stay , or 28-day mortality between the two groups , all of these were still shorter in the levosimendan group than the dobutamine group . the number of septic shock patients investigated in our study was small and the study period was relative brief , therefore , the risk of positive results in a study with numerous secondary variables has to be taken into account . second , our study did not show a statistically significant difference in 28-day mortality between the two groups and the difference was 5.2% , suggesting that the effect size was much lower than that assumed in the sample size calculation . post hoc sample size calculations showed that 1,305 patients per group would be required to show a statistically significant difference in 28-day mortality between the two groups . it is impossible to achieve a study like this in a single center ; a multi - centers investigation would be required in the future . finally , it is also indispensable to make clear the molecular biologic mechanisms of decreased hfabp in septic shock patients treated by levosimendan . the number of septic shock patients investigated in our study was small and the study period was relative brief , therefore , the risk of positive results in a study with numerous secondary variables has to be taken into account . second , our study did not show a statistically significant difference in 28-day mortality between the two groups and the difference was 5.2% , suggesting that the effect size was much lower than that assumed in the sample size calculation . post hoc sample size calculations showed that 1,305 patients per group would be required to show a statistically significant difference in 28-day mortality between the two groups . it is impossible to achieve a study like this in a single center ; a multi - centers investigation would be required in the future . finally , it is also indispensable to make clear the molecular biologic mechanisms of decreased hfabp in septic shock patients treated by levosimendan . compared with dobutamine , this study demonstrated levosimendan reduced the level of biomarkers of myocardial injury including hfabp , tni , and bnp , increased lvef , strengthened systolic function , and improved systemic hemodynamics significantly in patients with septic shock ; but there were no significant difference in duration of mechanical ventilation , length of stay in icu , or 28-day mortality between the two treatment groups . in conclusion , although levosimendan improved the biomarkers of myocardial injury and hemodynamic parameters , our study failed to show any improvement in clinically relevant outcomes like length of icu stay , duration of mechanical ventilation , and 28-day mortality . protein protein interactions are essential to the function of the ubiquitous xenobiotic - metabolizing cytochrome p450 monooxygenases , or cyps . classic studies have demonstrated the importance of cyp interactions with the accessory proteins cytochrome p450 reductase ( cpr ) and cytochrome b5 ( b5 ) for both electron transfer and allosteric modulation . however , it has recently become clear that cyp enzymes may participate in additional interactions with other protein partners , such as progesterone receptor membrane component 1 ( pgrmc1 ) , human serum albumin ( hsa ) , and even other cyp isoforms . these interactions have been shown to alter cyp function and , in some cases , lead to the production of toxic metabolites . in this review , we attempt to summarize the available evidence from the primary literature and offer some future perspectives on the study of noncanonical protein protein interactions with cytochrome p450 enzymes . cytochrome p450 reductase ( cpr ) was identified as the primary cyp electron transfer partner by the late 1960s . the term cytochrome p450 reductase is a bit of a misnomer , as this enzyme participates as a reducing partner in a number of different reactions , including those catalyzed by heme oxygenases i and ii , squalene monooxygenase , and certain fatty acid desaturases . initially characterized as a cytochrome c reductase , cpr was eventually found to be localized in the endoplasmic reticulum , where it functions as a redox partner with cyps that are also located there . cpr is a diflavin ( fmn , fad ) containing reductase that receives its electron - reducing equivalents from nadph . the cpr electron transfer protein shuttles two electrons to the cyp during the reaction cycle , with the second electron transfer thought to be the rate - limiting step . cpr is known to be post - translationally modified and has an apparent molecular weight between 76 and 80 kda , depending on post - translational modifications , the species - specific isoform , and detergent solubilization procedures . in addition to the fmn , fad , and nadph binding domains , cpr also contains a 60 amino acid hydrophobic n - terminal membrane anchoring region , which is essential for full activity with native redox partners , such as cytochrome p450 enzymes . a number of important crystal structures of cpr and several of its mutants have been produced , including those demonstrating important conformational changes that take place upon reduction and cyp binding . the first crystal structure of cpr was obtained using an n - terminally truncated sequence of the rat isoform . this structure demonstrated that the nadph fad binding domain also contained an important linker sequence that could help position the fmn and fad domains in close proximity for efficient electron transfer . the linker sequence distinguishes cpr from the closely related , single - domain ferrodoxin reductases . it is worthwhile to note that this particular n - terminally truncated construct was not able to support cyp reduction , even though it successfully reduced cytochrome c. more recent x - ray crystal structures have provided some insight into cyp the crystal structure of a cpr mutant with a four amino acid deletion in the hinge region between the fmn binding domain and the rest of the protein demonstrated remarkable flexibility in this region by crystallizing in three different conformations . although this particular mutant was not able to catalyze intramolecular electron transfer between the fad and fmn prosthetic groups , it was able to successfully reduce cyps , given enough reducing equivalents . the three different conformations observed suggest that cpr inherently possesses a high degree of conformational plasticity , especially in the c terminus of the hinge region , allowing it to efficiently interact with a variety of cyp isoforms and other enzymes . other structures obtained from protein x - ray crystallography and nmr have confirmed this conformational flexibility and the ability of cpr to transition between closed and open forms , which had also been predicted by computational molecular dynamics studies . another intriguing possibility is that subtle structural differences between cyp isoforms may regulate interaction with cpr and cytochrome b5 . seminal work examining structural and functional variation in the cpr gene has been conducted by both the masters and kranendonk groups , underscoring the importance of cpr in drug disposition and disease . several mutations in the gene encoding cpr are known to be associated with specific disease states , such as a rare form of congenital adrenal hyperplasia known as antley amino acid changes that are implicated in these conditions include y181d , a457h , y459h , v492e , and r616x . surprisingly , despite the large number of cyps present in the human genome ( 57 , excluding possible pseudogenes ; http://drnelson.uthsc.edu/hum.html ) , there is only one gene encoding cpr . additionally , the total cyp content of the human liver is estimated to be between 5- and 25-fold higher than that of cpr , suggesting differential expression . the fact that cyps and cpr interact in a 1:1 stoichiometry raises an unusual conundrum : exactly how do cyps and cpr interact to promote efficient oxidation of substrates ? from a structural perspective , it stands to reason that all of the different cyp isoforms must share a common functional cpr binding surface . indeed , several studies have proposed that the binding interaction between cyp and cpr takes place between the positively charged proximal surface near the cyp heme and the negatively charged fmn and fad / nadph domains of cpr , suggesting that their interaction is primarily driven by electrostatics . however , there is some controversy in this regard , as other studies have suggested that hydrophobic interactions are more predominant . these discrepancies may be the result of the different experimental approaches employed ( e.g. , x - ray crystallography vs site - directed mutagenesis and stopped - flow fluorescence spectroscopy ) . cpr complex is remarkably low , between 5 and 110 nm , depending on the particular isoform used and whether substrate is present , implying a very high - affinity interaction . upon initial examination , a tight - binding interaction may seem counterintuitive ; however , a rapid kon or koff rate would allow the system to work more efficiently due to swift equilibration . clearly , the interaction can be modulated by the presence of substrates and/or inhibitors , as well as other cyp isoforms , as noted below . another compounding factor is the variety of methods , often indirect , that have been used to determine cyp separate from its function as an electron transfer partner , cpr may also act as an allosteric effector to induce conformational changes in cyps that modulate the rate of electron transfer . a number of useful structural studies conducted with the bacterial model cyp isoform , cyp101 ( p450cam ) , have been useful to inform our understanding of how cpr might interact with mammalian cyps in order to modulate electron transfer . trans isomerization redox switch in cyp101 ( p450cam ) that is triggered by binding to the reduced form of cyp101 s cognate electron transfer partner , putidaredoxin ( pdx ) . these conformational changes account for most of the significant structural perturbations observed by nmr upon complex formation . conformational plasticity may be one mechanism by which the binding affinity of a cyp and its redox partner are modulated : if the subset of conformational states occupied by the reduced electron transfer protein is the same as that selected upon binding to a cognate cyp , then the entropic cost ( i.e. , free energy ) of binding would be lowered , resulting in a higher affinity . in a unique approach to examine cyp electron transfer partner interactions , hiruma and colleagues utilized paramagnetic nmr in conjunction with a site - specific lanthanide label to solve the solution structure of the cyp101pdx complex . this cyp101pdx complex structure was , in turn , validated by simultaneous determination of the x - ray crystal structure of the same complex . in contrast to reports of the interaction between mammalian cyps and cpr , here hydrophobic networks dominated the binding interface between the two proteins . the data suggested a minor conformational substate ( or substates ) that could represent an encounter complex between the two proteins , as postulated by an earlier study . additionally , the retrieved structural parameters were consistent with an electron transfer rate theoretically faster than that observed experimentally , suggesting the involvement of a gating process in electron transfer . similar allosterically induced conformational changes have also been observed with mammalian cyps and cytochrome b5 . taken together , these studies underscore the dynamic nature between cyps and their electron transfer partners , both from a functional standpoint as well as a structural one . seemingly small structural changes , such as a single amino acid substitution , could have dramatic functional consequences on the ability of cpr to efficiently deliver its electrons to a particular cyp isoform . recent studies have examined how genetic variation in the human cpr gene can manifest itself in the form of alterations in the isoform dependent cyp - mediated metabolism of certain substrates ( table 1 ) . interestingly , the effects of specific cpr mutations seem to be cyp isoform dependent , suggesting a unique interaction surface between individual cyp isoforms and cpr . in a study examining the effect of the 35 most common cpr mutations on the activity of cyp1a2 and cyp2c19 , miller and colleagues discovered that while most mutations either decreased the activity of both cyp1a2 and cyp2c19 , or eliminated activity all together ( most notably the a287p and r457h mutations ) , certain mutations enhanced cyp activity . in most cases , the enhancements were specific to the cyp isoform examined and the particular cpr mutation . for cyp1a2 , activity enhancement was observed with cpr mutants g213e , r406h , and a462 t . in the case of cyp2c19 , increased activity was reported for cpr mutants m263v and g504r . most surprisingly , the q153r cpr mutation , a rare disease - causing mutation that inhibits steroidogenic activity , increased the activity of both cyp1a2 and cyp2c19 to 144 and 284% , respectively . the authors rationalized these results by suggesting that many of the cpr mutants with reduced activity had decreased fad binding or lay in a region known to be important for cpr cyp contacts . the cpr gain - of - function mutants were more difficult to explain , but miller and colleagues suggest that , at least in the case of the q153r mutant , electron transfer rates to the cyp may be affected by the close proximity of glutamine residue 153 to the fmn moiety . the change in electrostatic charge in the q153r mutant may accelerate electron transfer from cpr to the cyp . whatever the mechanism involved , it is clear that there are important functional consequences for subtle alterations in the interactions between cpr and cyp isoforms . a follow - up study examined the effects of cpr mutations on the ability of cyp2d6 to metabolize different substrates . interestingly , for any particular cpr mutation , different effects were seen depending on the substrate used . the q153r cpr mutation mentioned above had a slight stimulatory effect on the cyp2d6-mediated metabolism of 2h-1-benzopyran-3-carbonitrile,7-(ethoxy - methoxy)-2-oxo-(9cl ) ( eomcc ) , yet it almost doubled the rate of metabolism of dextromethorphan . this observation may speak to the promiscuous nature of drug - metabolizing cyps and the multiple substrate binding sites within the active site . additional work with the cyp3a4 isoform also indicated that it was susceptible to functional variation due to cpr mutations . consistent with the previous findings for cyp2d6 and cyp1a2 , cyp3a4 activity is decreased in combination with most of the cpr mutants examined , but it is increased with the q153r mutant . this effect was again substrate - specific for the substrates testosterone and quinidine , and nominal activity was observed for midazolam and erythromycin oxidation . a study utilizing the full - length cyp and cpr mutant isoforms simultaneously expressed in insect sf9 cells via a baculovirus vector recapitulated the results obtained with the bacterially expressed isoforms ( referenced above ) . a result unique to this study was the observation that some of the reduced - function cpr mutants regained activity when coexpressed with cyp isoforms in the sf9 insect cells . this suggests that mutant cpr association with a cyp may help to stabilize the cpr protein into an active conformation , thereby rescuing activity . this is an intriguing possibility , given the postulate by pochapsky regarding the ability of a cyp to select the active conformation of a particular electron transfer partner . additional studies have pointed to the importance of the n - terminal hydrophobic anchoring sequence in supporting cyp - mediated oxidation . hayashi and colleagues found that an n - terminally truncated form of rat cpr failed to support cyp1a1-mediated 7-ethoxycoumarin o - deethylation , although it efficiently reduced cytochrome c and rat heme oxygenase i. furthermore , structural studies have demonstrated that crystallized , yet catalytically active , yeast cpr has a substantially different structure than the conventional n - terminally truncated catalytically inactive rat enzyme , reinforcing the importance of this sequence in supporting efficient electron transfer from cpr to cyp . cpr genetic variants may also have effects on different cyp isoform alleles as well . in a study examining the cyp2c9 alleles cyp2c9.1 , cyp2c9.2 , and cyp2c9.3 , tracy and colleagues found increased metabolic oxidation of the substrates diclofenac , flurbiprofen , and tolbutamide with four different mutant cpr enzymes tested ( q153r , a287p , r457h , and a503v ) . this was in contrast to the predominate effect seen previously with cyp1a2 , cyp2d6 , cyp2b6 , and cyp3a4 , where a decrease in activity was reported with these cpr mutants . the authors noted that since the effects they observed led to an increase in the vmax , but not a change in the km , it was most likely that the increase in activity observed was due to enhanced electron transfer and not an increase in cyp substrate affinity . variation in the cpr gene can lead directly to toxicity for certain drugs that are metabolized through reduction . the herbicide paraquat produces toxicity through the generation of superoxide anion during redox cycling . examined the effect of six common cpr mutations in patients with impaired steroidogenesis on the redox cycling of paraquat in a cho cell model system . in five out of the six cpr mutants examined , paraquat toxicity was diminished . although not explicitly confirmed in this study , the authors inferred that the mutants either affected the catalytic activity or expression levels of cpr , thereby reducing its ability to activate paraquat . in the c569y mutant , the observed paraquat toxicity was equivalent to that with the wild - type enzyme , implying a rescue of function with this particular mutation . the authors suggest that variation in the cpr gene may be a primary susceptibility determinant in paraquat toxicity . similarly , in a study investigating the toxicity of mitomycin c , wang et al . found that cpr mutations may be useful as biomarkers to predict the therapeutic response to this anticancer drug . mitomycin c is a common chemotherapeutic agent that is metabolically activated by either a one- or two - electron reduction step . cpr is the primary enzyme responsible for the one electron reduction , which produces the semiquinone anion radical that is thought cause damage to cellular dna . of six cpr mutants that were examined ( y181d , a287p , r457h , v492e , c569y , and v608f ) , cellular toxicity of mitomycin c was decreased in all with the exception of the c569y mutant , demonstrating that a high degree of cpr functional activity is needed to activate mitomycin c. in particular , the v608f and y181d mutants lacked any ability to activate mitomycin c , presumably due to altered association with the flavin moiety . in addition to mitomycin c , the impact of cpr variants has also been examined with tacrolimus , warfarin , and other drugs , illustrating the importance of this particular enzyme in drug metabolism and elimination . cytochrome b5 ( b5 ) is a small ( 17 kda ) heme - containing integral membrane protein located on the cytosolic side of the endoplasmic reticulum and was originally identified in 1950 in silkworm larva . although the majority of b5 is membrane - bound , a certain fraction is present as a soluble , cytosolic form in erythrocytes , where it catalyzes the reduction of methemoglobin . although mutations in the b5 gene itself are quite rare , a few have been traced to the congenital abnormality of 17,20-lyase deficiency . despite the fact that it can act as an electron donor to cyp enzymes , b5 is primarily involved in lipid biosynthesis , delivering electrons to microsomal desaturases that synthesize steroids , fatty acids , and plasmalogens . in the early 1970s , estabrook and mannering first demonstrated that b5 stimulated the metabolism of several cyp substrates . however , at this time , the functional role b5 played in cyp - catalyzed oxidation was still somewhat unclear . early reports suggested that it could be involved in delivery of the second electron in the cyp catalytic cycle , thereby accelerating the rate - limiting step in cyp - mediated catalysis . remarkably , a more contemporary report from the wolf laboratory in a hepatic cpr deletion mouse model established that b5 and b5 reductase can act as efficient cyp electron donors , without the need of cpr . despite its role in electron transfer , the effects seen from b5 are highly variable and dependent on the particular reaction conditions used , as addition of b5 can either inhibit or have no effect on some reactions rather than stimulate them . furthermore , several studies have demonstrated that addition of apo ( heme free ) b5 can stimulate the activities of many cyps , including cyp3a4 , cyp2c9 , and cyp17 . it has been postulated that , in certain cases at least , b5 exerts the majority of its effects in the absence of electron transfer . it is still not completely clear if b5 functions solely as an electron donor , allosteric modulator , electron sink , or an uncoupling inhibitor . it is entirely plausible that its function may be contextual , depending on the particular isoform / substrate combination with which it is interacting . a number of elegant studies over the years have established that electrostatic interactions are the basis for formation of the cyp waskell and colleagues mapped out the binding interface between cyp2b4 and b5 through extensive site - directed mutagenesis of both proteins and identified r122 , r126 , r133 , k139 , and k433 of cyp2b4 as residues critical to their interaction . recently , a number of structural and dynamic 2d nmr studies have provided additional insight into the cyp embedded in lipid bicells , ramamoorthy , waskell , and colleagues were able to take advantage of rotating frame separated local field ( slf ) nmr . the authors employed two - dimensional proton evolved local - field ( 2d pelf ) pulse sequences to accurately measure a broad range of heteronuclear dipolar couplings , allowing for a complete mapping of b5 protein dynamics in a native lipid bilayer environment . notably , the authors discovered that the hydrophobic n - terminal helix of b5 precessed in a cone with an approximate 8 fluctuation in the tilt of the helix , which is estimated to be 13 , suggesting that the lipid - embedded hydrophobic n - terminus is more mobile than originally thought . this added flexibility may allow this region of the protein to interact with a diverse variety of cyp isoforms . there is a growing appreciation of how inherent protein flexibility can allow proteins to adopt non - native states , which may allow them to gain new functions or interact with different protein partners . , ramamoorthy and colleagues utilized a modified inept technique , dipolar enhanced polarization transfer ( drept ) , to detect an increased range of residual dipolar couplings from isotopically labeled b5 that allowed them to characterize the amino acid side chain motions of b5 while embedded in lipid bicells . two - dimensional nmr methodologies have also been useful in providing explanations for the specific rate enhancements seen with certain cyp substrate pairs . the bifunctional steroidogenic enzyme cyp17a1 performs both steroid hydroxylation , which is unaffected by b5 , and an androgen - forming lyase reaction that is enhanced 10-fold in its presence . work by the scott group examining the interactions between an isotopically labeled b5 construct and cyp17a1 led to the observation that the cyp17a1b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the cyp17a1 active site than when the lyase substrate 17-hydroxypregnenolone is bound . this suggests that , at least in this case , the b5 reaction rate enhancement may be primarily driven by substrate affinity . in a follow - up study , in which cyp17a1 was isotopically labeled and the b5 construct was unlabeled , titration of b5 into cyp17a1pregnenolone complex induced a set of conformational substates closely resembling those of the cyp17a117-hydroxypregnenolone complex without b5 . this result implied that b5 may also be able to allosterically induce catalytically productive conformations in cyp17a1 , even in the absence of substrate . these studies as well as others have culminated in a complete structural and catalytic model for the cyp ahuja et al . determined the structure of full - length , membrane bound b5 in the presence of cyp2b4 . this structure , determined for the first time in the presence of a phospholipid bilayer , confirmed the electrostatic nature of the cyp b5 interaction , revealing a large number of charge pairings between surface residues on cyp2b4 and b5 that facilitate the interaction between the two proteins . however , it also hinted at the importance of hydrophobic interactions for the complex that are mediated by the phospholipid bilayer . interestingly , the authors described an increase in the affinity between cyp2b4 and b5 in the presence of a small molecule substrate or inhibitor , similar to that observed by estrada et al . for the cyp17a1b5 interaction . additionally , their results suggest a pathway for electron transfer between cyp2b4 and b5 mediated through a salt bridge with arg125 of cyp2b4 and the heme propionates of b5 . this study illustrates the power of 2d nmr techniques to obtain detailed structural data for the interactions between two protein partners . finn and colleagues created a liver - specific b5 conditional knockout mouse in order to better define the functional importance of the cyp b5 interaction.in vitro metabolism studies demonstrated the complete lack of nadh - mediated metabolism and a 50 to 90% decrease in nadph - mediated metabolism for most of the drug substrates examined . additionally , a dramatic increase in the half - life for these particular drug substrates was noted , thus establishing that b5 is a major contributor to the in vivo metabolism of many drugs and xenobiotics , albeit in a substrate- and isoform - selective manner . a follow - up study demonstrated altered clearance of several anticancer agents in the same knockout mouse model . therefore , mutant b5 alleles that are deficient in their ability to interact with , and transfer electrons to , hepatic cyps could lead to altered clearance rates for certain drugs , which may contribute to drug - induced toxicities . furthermore , varying levels of b5 function important for drug oxidation can also produce altered metabolite ratios , potentially increasing the production of toxic products in certain instances . in a study conducted by the hollenberg group , b5 was determined to be essential for the formation of protein adducts to cyp3a5 from metabolic activation of the contraceptive agent 17-ethynylestradiol . in the absence of b5 , the reactive 17-oxirene - related species was not formed , leading to cyp enzyme protection . an additional concern from a toxicological point of view is the potential for the generation of reactive oxygen species ( ros ) due to increased uncoupling . indeed , using a reconstituted system containing both lipid and cpr , b5 has been shown to generate superoxide and hydroxyl radicals in the presence of cr(vi ) , leading to double - stranded dna breaks in vitro.(109,110 ) the fact that cpr was needed to observe the effect suggests the importance of the interaction between b5 and cpr to generate these ros . additionally , ros generated by b5 have been demonstrated to increase lipid peroxidation in reconstituted biological membranes , which may in turn lead to cellular damage and death . therefore , there are multiple mechanisms through which altered b5 levels and/or function can exert a toxicological effect . surprisingly , cyps also exhibit protein protein interactions with other cyps . in the early 1990s , research by coon , davydov , and others demonstrated that cyps can form homomeric complexes . work in the ensuing 2 decades has established that multiple cyp isoforms can interact with one another in both homomeric and heteromeric complexes and that these complexes can often have significant effects on cyp - mediated oxidation of substrates ( for recent reviews , please see references ( 117 ) and ( 118 ) ) . for simplicity , we have grouped the cyp cyp interactions into those described by homomeric ( two or more molecules of the same isoform ) and heteromeric ( two or more molecules of different isoforms ) . pioneering studies of homomeric cyp cyp interactions were conducted by davydov in the early to mid-1990s , examining the response of cyp2b4 to pressure induced conformational changes . davydov and his colleagues discovered that approximately 35% of the ferrous cyp2b4 co - bound complex was refractory to conversion into the inactive p420 form upon application of pressures in excess of 2 kbar . however , this refractory population was reduced to less than 5% upon addition of 0.2% triton n-101 , a detergent that created conditions known to produce monomers . these results implied that a significant portion of active cyp ( i.e. , cyp able to be reduced and bind co ) was present in the oligomeric state . later work by this same group would demonstrate that sodium dithionite or cpr induced reduction kinetics in cyp3a4 are multiphasic under conditions favoring cyp3a4 oligomers but monophasic under conditions favoring monomers . these studies are suggestive of active cyp arranged in homomeric oligomers in the phospholipid bilayer . confirmation of a direct physical interaction between individual cyps in a live cell membrane was achieved in seminal studies by kemper and colleagues using fluorescently labeled cyp2e1 and cyp2c2 in cells transfected with murine cyp cdna . they found that while fluorescence resonance energy transfer ( fret ) occurred between individual cyp2c2 molecules in a membrane , it could not be detected between cyp2e1 monomers , representing a homomeric self - association with cyp2c2 but , interestingly , not cyp2e1 . the cyp2c2 dimers were later confirmed to exist in murine hepatocyte endoplasmic reticulum membranes , indicating the in vivo relevance of such associations . taken together , these studies laid the groundwork for analysis of the functional consequences of cyp cyp homomeric interactions . in an elegant kinetic study conducted by jamakhandi et al . , the authors found that metabolism data for cyp2e1 fit best to either a quaternary ( cyp cpr cyp cpr ) , trimeric ( cyp cyp cpr ) , or dimeric ( cyp cyp ) complex model where only the binary ( cyp cpr ) complex was active . the authors found that simply varying the individual concentrations of cyp or cpr in a reconstitution system produced contradictory results , which did not correspond to the simplest functional ( 1:1 ) complex . simultaneously , job titration analysis revealed an asymmetric plot , indicative of higher - order molecular complexes . in an attempt to identify the catalytically relevant species , both cyp and cpr concentrations were titrated simultaneously , resulting in a two - dimensional data surface . when the data were globally fit to 32 different candidate models ( including binary , ternary , and quaternary cyp cpr complexes ) , it revealed that the most likely model was one composed of the binary complex ( cyp cpr ) , the quaternary complex ( cyp cpr cyp cpr ) , and the homodimer ( cyp cyp ) . a second , although less likely , possibility was a model that involved a weakly bound ternary complex ( cyp - cyp interestingly , the results implied that the binary complex was the only catalytically active state . therefore , in this case , even though cyp2e1 was shown to be capable of forming cyp cyp complexes that could have an effect on catalytic activity , the binary cyp more recently , a comprehensive study by the backes group examining the effect of homomeric cyp complex formation on the activity of cyp1a2 , cyp2b4 , and cyp2e1 found that , at least in the case of cyp1a2 , cyp cyp homomeric complexes contribute to altered catalytic activity seen with this enzyme under conditions that would promote dimer formation . to understand how cyp homomeric complexes might affect function , the catalytic activities of several individual cyps , including cyp2b4 , cyp2e1 , and cyp1a2 , were assessed in reconstituted systems as a function of cyp concentration . interestingly , although oxidation mediated by cyp2b4 as a function of cyp cpr best fit to a simple hyperbolic michaelis menten equation , cyp2e1 and cyp1a2 demonstrated atypical ( non - michaelis menten ) kinetics that fit best to a sigmoidal curve , indicating a high degree of cooperativity with these particular isoforms . this non - michaelis menten kinetic behavior could be converted back to a hyperbola simply by increasing the ionic strength of the buffer , suggesting that the cyp cyp complex in the membrane using a variety of biophysical techniques , including chemical cross - linking and bioluminescence resonance energy transfer ( bret ) . in yet another study , cyp interaction was found to affect enzymatic function by a direct modulation of allosteric ( non - michaelis menten ) enzyme kinetics . both homotropic and heterotropic cooperativity have long been known to occur with hepatic drug - metabolizing cyp isoforms , particularly cyp3a4 . recently , davydov and colleagues reported that stimulation of cyp3a4-mediated oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin ( 7-bfc ) by -naphthoflavone in human liver microsomes ( hlm ) was highly dependent on the degree of cyp3a4 oligomerization . the amount of enzyme activation seen was directly correlated with the surface density of the enzyme . while no activation was detectable at high lipid / cyp ratios ( 750 ) , activation reached greater than 225% at a lipid / cyp ratio of 140:1 . this suggests that allosteric activation requires at least some degree of homomeric cyp cyp interaction . cyp complexes , at least in the dimeric form , it is still not entirely clear what role these complexes may play in vivo in the detoxification and elimination of xenobiotic agents , especially under conditions of cyp induction or endoplasmic reticulum ( er ) stress . one suggested physiological function basis for the formation of cyp cyp complexes is their ability to prompt a hypothetical functional allosteric mechanism future studies with relevant in vivo model systems may shed some light in this area . the identification of the existence of homomeric cyp complexes has led to interest in the possibility of heteromeric cyp complexes as well . using the same methodology that revealed homomeric interactions between individual cyp2c2 molecules , kemper and colleagues identified additional heteromeric interactions between cyp2c2 and cyp2e1 . this implied that differences in homo- and heteromeric oligomerization state between isoforms may help explain some of the relative differences seen in activity . indeed , this was found to be the case in complexes formed between cyp1a2 and cyp2b4 in reconstituted systems . in a reconstituted system containing cpr , lipid , and both cyp1a2 and cyp2b4 enzymes , a small increase in catalytic activity for the cyp2b4 substrate benzphetamine was detected . however , activity against another cyp2b4 substrate , 7-pentoxyresorufin - o - dealkylation ( prod ) , was dramatically inhibited . inhibition of prod was also dependent on cpr levels , with the inhibitory effect being more pronounced at subsaturating cpr concentrations , suggesting competition for reductase binding between the two isoforms . in a follow - up study , backes and colleagues determined that it was likely that a ternary complex containing cpr , cyp1a2 , and cyp2b4 was formed , for cyp2b4 activity was dramatically reduced at subsaturating reductase concentrations . this result suggested an alternative model where , under substoichiometric conditions , cpr is specifically bound to cyp1a2 and at higher cpr concentrations , reductase binding to cyp1a2 becomes saturated , resulting in the formation of a quaternary complex in which cpr binds to both cyp1a2 and cyp2b4 enzymes . in a more recent study examining the interaction between cyp2c9 and cyp2c19 in reconstituted systems , kupfer and colleagues found evidence of a metabolic interaction between even these highly related cyp isoforms . previously , using in vitro systems reconstituted with purified enzyme , cyp2c19 was the most active isoform for methoxychlor - o - demethylation . however , in hlm , cyp2c9 performed the bulk of methoxychlor - o - demethylation . interestingly , while an equimolar ratio of cpr to cyp2c9 supported maximal rates of methoxychlor - o - demethylation , the rate of methoxychlor - o - demethylation by cyp2c19 was not fully saturated in the same system ; even when reconstituted with a 9-fold molar excess of cpr , cyp2c19 methoxychlor - o - demethylation and s - mephenytoin hydroxylation kinetics were not fully saturated ( as compared with that in hlm ) . however , when a binary reconstitution system was prepared by mixing cyp2c9 and cyp2c19 enzymes , methoxychlor - o - demethylation and s - mephenytoin hydroxylation by cyp2c19 were inhibited dramatically , with the amount of inhibition dependent on the amount of cpr and substrate used , yet increasing concentrations of cpr were generally able to overcome the inhibitory effects . by contrast , in the incubation containing only cyp2c9 , diclofenac hydroxylation was activated by the presence of cyp2c19 , thus demonstrating that cyp cyp interactions can modulate the catalytic rates of a variety of oxidation reactions but that this modulation is highly dependent on the substrate undergoing metabolism , with some substrates activating metabolism and others inhibiting it . surprisingly , subramanian and colleagues found that while addition of cyp2d6 to a reconstituted incubation of cyp2c9 and ( s)-flurbiprofen increased the ks by 20-fold , it had no effect on the km , but it decreased the vmax by 50% . one possible explanation for this discrepancy is that affinity constants determined by heme spin state ( i.e. , optical spectrum ) perturbation may mask total ligand binding because they measure only substrate interactions that perturb the heme iron . it is possible that , in the presence of an additional cyp isoform , the spin state shift may be less complete , thereby not reflecting the effects of additional ligand binding . the authors additionally noted that the effects on metabolism were dependent on the order of addition of the enzymatic constituents , with the greatest rate enhancements observed when cpr was added before the second cyp , again suggesting a competition for reductase binding . in yet another study , yamazaki et al . discovered similar types of enzymatic stimulation between cyp1a2 and other isoforms , including cyp3a4 , cyp2c9 , and cyp2d6 , which indicate a more generalizable phenomenon among the hepatic cyps . as more substantial biophysical and functional evidence has accrued demonstrating heteromeric interaction between individual cyp isoforms , attention has shifted to structurally defining the mechanism of this interaction . two different studies conducted in 2010 by the backes and tracy laboratories were instructive in this regard . the backes group set out to answer the question of whether two different cyp isoforms need to be located in the same membrane to functionally interact , thereby potentially excluding long - range allosteric effects . their experimental system consisted of incorporating cyp1a2 and cyp2b4 into vesicles with cpr individually and together as a cyp cyp complex . if the two cyps functionally interacted in the same membrane , then metabolic stimulation ( or inhibition ) would be observed only when both cyps were present in the same vesicle , and a mixture of both the individual cyp1a2cpr and cyp2b4cpr vesicles would simply produce an additive effect . indeed , this is exactly what the authors observed prior to confirming this functional interaction between these two cyps with cross - linking and coimmunoprecipitation experiments . work from the tracy lab examined the effect of the hydrophobic n - terminal anchor sequence on mediating the interaction between cyp2c9 and cyp3a4 . using a reconstituted enzyme system containing both cyp2c9 and cyp3a4 , the authors demonstrated that cyp2c9 mediated ( s)-naproxen metabolism was inhibited as much as 80% by the presence of cyp3a4 , while the km remained unchanged . the degree of inhibition was directly proportional to the cyp3a4 concentration and indirectly proportional to the cpr concentration , suggesting a competition for reductase . oddly , cyp2c9 did not seem to alter cyp3a4-mediated testosterone metabolism . in order to study the role of the hydrophobic n - terminal anchor sequence , the experiments were performed with both n - terminally truncated and full - length cyp isoforms . there was no evidence for metabolic inhibition when the full - length cyp3a4 and cyp2c9 isoforms were incubated in the presence of the truncated form of the other cyp isoform , but evidence for inhibition was found only when both full length forms were present . this indicated that interaction between the hydrophobic n - termini of the two cyp isoforms was required for functional inhibition . it is possible that , in this context , the hydrophobic n - termini may orient the cyps in the membrane . finally , the authors conducted coimmunoprecipitation studies to demonstrate that the two cyps were directly interacting in the membrane . it has now been firmly established that cyp s form both homomeric and heteromeric complexes with functional consequences . the difficult work of defining how these complexes interact structurally and what their in vivo roles may be now lies ahead . of all the protein protein interactions in which cyps have been documented to participate , perhaps the most enigmatic is that with nonenzymatic proteins , including the progesterone receptor membrane component 1 ( pgrmc1 ) and bovine and human serum albumin ( bsa / hsa ) . although these proteins do not provide reducing equivalents to the cyp catalytic cycle , they are still capable of modulating cyp - mediated metabolism . presently , these mechanisms remain to be fully elucidated ; intriguingly , they may involve direct delivery of a drug substrate to the particular cyp . below , we provide a brief summary of what is currently known regarding these cyp protein protein interactions . despite its rather precise identification , the progesterone receptor membrane component 1 is a somewhat misleading name , as it neither binds progesterone directly nor does it share any homology with progesterone receptors . in contrast , pgrmc1 shares a high degree of structural homology with cytochrome b5 . pgrmc1 has been demonstrated to either increase or decrease the rate of cyp - mediated metabolism , depending on the cyp pgrmc1 also seems to play a role in the development of certain cancers , as recent reports suggest that it may be the inscrutable receptor , a known biomarker of tumor cell proliferation . in mammals , there exist two additional pgrmc1 family isoforms , prgmc2/dg6 and neudesin , although it is not known if they also interact with cyp enzymes . prgmc1 is highly expressed in the human liver and typically colocalized in the smooth endoplasmic reticulum ( ser ) with cyp proteins . it has been shown to interact directly with cyp enzymes , including cyp3a4 , cyp2c9 , cyp7a1 , cyp21a2 , and cyp51 . pgrmc1 activates progesterone oxidation mediated by cyp21a2 and is essential for cyp51 activity in the cholesterol synthesis pathway . min and colleagues reported that the coexpression of pgrmc1 with cyp21a1 enhanced progesterone 21-hydroxylation in cos-7 cells , while a heme - deficient pgrmc1 mutant had no effect . in an experiment with the yeast homologue , dap1 , hugues et al . showed that both cyp51a1 and cyp61a1 can be positively regulated by the presence of dap1 . remarkably , prgmc1 has been revealed to exhibit isoform - selective effects in its interaction with cyps . oda found that pgrmc1 generally reduced cyp activities , either through decreased vmax and km values ( in the case of cyp3a4 ) or simply through a decreased vmax ( in the case of cyp2c9 ) ; however , in the case of cyp2e1 , it had no effect . coimmunoprecipitation studies suggested that the mechanism behind the differential effects may be differences in binding affinity between pgrmc1 and individual cyp isoforms . szczesna - skorupa and kemper found similar results in their studies of the interactions of pgrmc1 with cyp2c2 , cyp2c8 , and cyp3a4 in transfected hepg2 cells . in each case , pgrmc1 downregulated cyp activities , which could be rescued by increased expression of cpr . pgrmc1 was found to have a high affinity for cpr , but only in the absence of cyp2c2 , indicating competition between pgrmc1 and cyp2c2 for binding to cpr . in contrast to what was observed with the drug - metabolizing cyp isoforms , cyp51 activity was reduced with decreasing levels of pgrmc1 , indicating that a separate mechanism may be in place for the interaction of pgrmc1 with cyps involved in steroid synthesis . the mechanism of cyp activation or inhibition by pgrmc1 is highly debatable ; however , it is conceivable that it may involve drug delivery or sequestration , as a hydrophobic binding pocket has been identified on pgrmc1 . in any case , much work remains to elucidate the structural and functional details of the cyp human serum albumin ( hsa ) is a 65 kda monomeric , slightly basic globular protein that circulates as a major component of blood plasma and is the most abundant protein in the human body . hsa functions primarily as a carrier for steroids , fatty acids , and thyroid hormone . additionally , it plays a role in stabilizing osmotic pressure by regulating extracellular fluid volume . hsa binds a large variety of drugs and xenobiotic agents and has a major impact on their pharmacokinetics and disposition . under normal physiological circumstances , hsa however , it is synthesized in the liver and is present in the ser of hepatocytes , so it is conceivable that hsa could come into direct contact with cyp isoforms . moreover , the extent of drug or toxin protein binding has long been known to effect in vitro - to - in vivo extrapolations of disposition . therefore , in an effort to improve in vitro - to - in vivo predictions , researchers began to add either hsa or bovine serum albumin ( bsa , a cheaper and more well - characterized substitute ) to both microsomal incubations and recombinant baculovirus - expressed enzymes . this accomplished the desired effect of improving pharmacokinetic predictions by increasing the in vitro intrinsic clearance rate , clint ( vmax / km ) to more closely match what is seen in vivo . this effect is often mediated through a decrease in the substrate km , and it has been observed for a number of different cyp isoform / drug combinations , including cyp2c8 , cyp2c9 , and cyp1a2 . however , for some cyp isoforms , including cyp2c19 and cyp2d6 , bsa was found to be inhibitory . established that the degree of activation or inhibition observed was dependent on the concentration of bsa used , with lower concentrations activating and higher concentrations being inhibitory . in addition , bsa s cyp activation / inhibition profile was demonstrated to deviate from that observed with hsa , indicating the importance of the albumin source for comparison studies and in vitro - to - in vivo extrapolations . a number of theories have been proposed to account for the effect seen with albumin , including direct drug delivery , allosterically induced conformational change , and inhibitory long - chain unsaturated fatty acid sequestration . although no single theory fits the data precisely , recent evidence seems to lend credence to long - chain unsaturated fatty acid sequestration . of course , because none of these theories are mutually exclusive , it may be that multiple mechanisms are involved rather than a single predominant mechanism with any particular drug / cyp isoform . lastly , other proteins related to albumin , such as human intestinal fatty acid binding protein ( ifabp ) , have also been demonstrated to improve in vitro - to - in vivo predictions based on microsomal incubations , suggesting a more generalizable phenomena with this class of proteins . in addition to clearly defining the particular mechanisms of albumin cyp interaction , future work in this area will also need to delineate exactly which members of this protein family interact with individual cyp isoforms . protein interaction will no doubt further enhance our ability to correlate in vitro metabolism to that observed in vivo . the cyp pathway of oxidative transformation of drugs and other xenobiotics is critical for the elimination of toxicity in the host organism . proper functioning of this pathway relies on effective formation of protein protein complexes among the cyps , their electron transfer partners , and other proteins that can influence their function . mutations in either the cyp or an interacting protein that effect the structure or stability of the cyp or its protein partner can lead to deficiencies or alternative pathways of biotransformation , which in turn may produce toxic metabolites . therefore , a comprehensive understanding of the many different types and functions of protein protein interactions that cyps undergo is necessary to predict and treat such toxicities . new techniques protein interactions that are beginning to be developed will be a boon for research in this area . additionally , from a pharmacological point of view , modulating protein protein interactions may provide an avenue for therapeutic intervention . conceivably , cyps might also interact with phase ii , or drug conjugating , enzymes such as glucuronidases , sulfotransferases , or gluthione - s - transferases to modulate their activity as well . protein interactions increases , we will not only improve our ability to predict and prevent toxic cyp drug interactions but also gain a new appreciation for the many unique attributes of this versatile enzyme family .

backgroundwe aimed to investigate the effect of levosimendan on biomarkers of myocardial injury and systemic hemodynamics in patients with septic shock.material/methodsafter achieving normovolemia and a mean arterial pressure of at least 65 mmhg , 38 septic shock patients with low cardiac output ( left ventricular ejective fraction ) , levf 45% ) were randomly divided into two groups : levosimendan dobutamine . patients in the levosimendan and dobutamine groups were maintained with intravenous infusion of levosimendan ( 0.2 g / kg / minute ) and dobutamine ( 5 g / kg / minute ) for 24 hours respectively . during treatment we monitored hemodynamics and lvef , and measured levels of heart - type fatty acid binding protein ( hfabp ) , troponin i ( tni ) , and brain natriuretic peptide(bnp ) . in addition , the length of mechanical ventilation , intensive care unit ( icu ) stay , hospital stay , and 28-day mortality were compared between the two groups.resultsthe levosimendan group and the dobutamine group were well matched with respect to age ( years , 55.41 7.5 versus 50.213.6 ) and gender ( males , 68.4% versus 57.9% ) . levosimendan - treated patients had higher stroke volume index ( svi ) , cardiac index ( ci ) , lvef , and left ventricular stroke work index ( lvswi ) , and lower extravascular lung water index ( evlwi ) compared to dobutamine - treated patients ( p<0.05 ) . hfabp , tni , and bnp in the levosimendan group were less than in the dobutamine group ( p<0.05 ) . there was no difference in the mechanical ventilation time , length of stay in icu and hospital , and 28-day mortality between the two groups.conclusionscompared with dobutamine , levosimendan reduces biomarkers of myocardial injury and improves systemic hemodynamics in patients with septic shock . however , it does not reduce the days on mechanical ventilation , length of stay in icu and hospital , or 28-day mortality . through their unique oxidative chemistry , cytochrome p450 monooxygenases ( cyps ) catalyze the elimination of most drugs and toxins from the human body . protein protein interactions play a critical role in this process . historically , the study of cyp protein interactions has focused on their electron transfer partners and allosteric mediators , cytochrome p450 reductase and cytochrome b5 . however , cyps can bind other proteins that also affect cyp function . some examples include the progesterone receptor membrane component 1 , damage resistance protein 1 , human and bovine serum albumin , and intestinal fatty acid binding protein , in addition to other cyp isoforms . furthermore , disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites . in this review , we summarize the available evidence for cyp protein protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein protein interactions with cyp enzymes .

methods and any associated references are available in the online version of the paper at http://www.nature.com / naturemethods/. difference in optimal collision energies for identification and quantification supplementary note real - time filtering supplementary table 1 summary of quantification and identification results supplementary protocol implementing quantmode on commercial mass spectrometers with the improvement of diagnostic imaging , the incidence of uia diagnosis is increasing , along with the national cost containment pressures associated with treatment and management . economic considerations are important in the cost - benefit evaluation of treatments , and influence healthcare coverage , reimbursement , and policy . while many studies have already compared the treatment modalities for intracranial aneurysms , surgical clipping and endovascular coiling , in terms of the procedural invasiveness , clinical and functional outcomes , and durability , few studies have investigated the economic costs for such treatments23713 ) . plus , treatment costs show distinct disparities between countries313).during the process of informed consent for clipping or coiling , uia patients are also concerned about the cost implications . therefore , the authors performed a cost comparison , and investigated the principal cost determinants of uia treatment in south korea . this study conducted a retrospective review of data from a series of consecutive patients who underwent surgical clipping or endovascular coiling of a uia at the authors ' institution between january 2011 and may 2014 . the exclusion criteria included a recent ( < 1 month ) subarachnoid hemorrhage caused by a concomitant aneurysm , giant aneurysm with a diameter > 2.5 cm , poor preoperative neurological state affecting postoperative management , and another disease or medical condition significantly increasing the hospital costs . the medical records were reviewed to obtain relevant clinical information , and all the radiological data were obtained using an electronic picture archiving and communication system . the treatment decision , surgical coiling versus endovascular coiling , was made primarily based on the findings of the digital subtraction angiography . surgical treatment was favored over endovascular treatment for patients with the following findings : 1 ) difficult navigation of the microcatheter into the aneurysm19 ) , 2 ) very small ( <3 mm ) aneurysm149 ) , 3 ) complex and wide - neck aneurysm requiring y or x stent - assisted coiling610 ) , 4 ) aneurysm with an arterial branch incorporated into the sac58 ) , 5 ) fusiform or complex aneurysmal configuration , or 6 ) aneurysmal compression of an adjacent cranial nerve11 ) , as long as the patients had no problems related to surgical accessibility or comorbidity . all the patients completed an angiographic examination , chest x - ray , and laboratory tests as a preoperative evaluation before admission , and were admitted to hospital one day before the surgical or endovascular procedure . the operative and perioperative management procedures were uniformly applied to each uia patient . for the patients who underwent surgical clipping , a postoperative computed tomography ( ct ) and ct angiography another ct scan was performed on the day of discharge to check for the occurrence of any subdural hygromas . for most surgical patients , the length of the hospital stay was 3 - 6 days . in the case of elderly patients , a longer hospital stay was usually required due to fatigue and general weakness . meanwhile , for the patients who underwent endovascular coiling , diffusion - weighted magnetic resonance imaging was commonly performed on day 1 following the procedure to evaluate any procedure - related thromboembolic phenomena . the length of the hospital stay after coiling was 3 - 4 days for most patients , irrespective of the patient 's age . the treatment costs for each patient were calculated based on the expenses incurred during the time spent as an inpatient for aneurysm treatment , including the operative and postoperative costs . the operative costs covered the neurosurgical or endovascular procedure , anesthesia , and all the materials ( e.g. , aneurysm clip , endovascular coil , and other surgical supplies ) , while the perioperative costs included the costs for the bed occupancy in the intensive care unit and regular hospital ward , radiological imaging studies , laboratory tests , and all the medications . the costs affected by patient selection ( e.g. , bed occupancy in the regular hospital ward ) were made uniform to the basic lowest cost . all the costs are all presented in korean won and us dollars based on an international conversion rate of 1100=$1 . the statistical analyses were performed using an spss software package ( version 18.0 for windows ; spss inc . , chicago , il , usa ) . plus , scientific data analysis and graphing software ( sigmaplot , version 10.0 ; systat software inc . , chicago , il , usa ) was applied for the statistical linear regression analysis . an independent t - test was used for the mean comparison between the surgical and endovascular groups . the correlation between the aneurysm diameter and the total hospital costs was assessed using a pearson correlation analysis . this study conducted a retrospective review of data from a series of consecutive patients who underwent surgical clipping or endovascular coiling of a uia at the authors ' institution between january 2011 and may 2014 . the exclusion criteria included a recent ( < 1 month ) subarachnoid hemorrhage caused by a concomitant aneurysm , giant aneurysm with a diameter > 2.5 cm , poor preoperative neurological state affecting postoperative management , and another disease or medical condition significantly increasing the hospital costs . the medical records were reviewed to obtain relevant clinical information , and all the radiological data were obtained using an electronic picture archiving and communication system . the treatment decision , surgical coiling versus endovascular coiling , was made primarily based on the findings of the digital subtraction angiography . surgical treatment was favored over endovascular treatment for patients with the following findings : 1 ) difficult navigation of the microcatheter into the aneurysm19 ) , 2 ) very small ( <3 mm ) aneurysm149 ) , 3 ) complex and wide - neck aneurysm requiring y or x stent - assisted coiling610 ) , 4 ) aneurysm with an arterial branch incorporated into the sac58 ) , 5 ) fusiform or complex aneurysmal configuration , or 6 ) aneurysmal compression of an adjacent cranial nerve11 ) , as long as the patients had no problems related to surgical accessibility or comorbidity . all the patients completed an angiographic examination , chest x - ray , and laboratory tests as a preoperative evaluation before admission , and were admitted to hospital one day before the surgical or endovascular procedure . the operative and perioperative management procedures were uniformly applied to each uia patient . for the patients who underwent surgical clipping , a postoperative computed tomography ( ct ) and ct angiography another ct scan was performed on the day of discharge to check for the occurrence of any subdural hygromas . for most surgical patients , the length of the hospital stay was 3 - 6 days . in the case of elderly patients , a longer hospital stay was usually required due to fatigue and general weakness . meanwhile , for the patients who underwent endovascular coiling , diffusion - weighted magnetic resonance imaging was commonly performed on day 1 following the procedure to evaluate any procedure - related thromboembolic phenomena . the length of the hospital stay after coiling was 3 - 4 days for most patients , irrespective of the patient 's age . the treatment costs for each patient were calculated based on the expenses incurred during the time spent as an inpatient for aneurysm treatment , including the operative and postoperative costs . the operative costs covered the neurosurgical or endovascular procedure , anesthesia , and all the materials ( e.g. , aneurysm clip , endovascular coil , and other surgical supplies ) , while the perioperative costs included the costs for the bed occupancy in the intensive care unit and regular hospital ward , radiological imaging studies , laboratory tests , and all the medications . the costs affected by patient selection ( e.g. , bed occupancy in the regular hospital ward ) were made uniform to the basic lowest cost . all the costs are all presented in korean won and us dollars based on an international conversion rate of 1100=$1 . the statistical analyses were performed using an spss software package ( version 18.0 for windows ; spss inc . , chicago , il , usa ) . plus , scientific data analysis and graphing software ( sigmaplot , version 10.0 ; systat software inc . , chicago , il , usa ) was applied for the statistical linear regression analysis . an independent t - test was used for the mean comparison between the surgical and endovascular groups . the correlation between the aneurysm diameter and the total hospital costs was assessed using a pearson correlation analysis . surgical clipping was applied to 222 patients , where a single aneurysm was clipped in 188 patients and 2 - 3 multiple aneurysms were clipped in 34 patients . while most patients underwent a superciliary keyhole approach ( n=180 , 81.1% ) , the others experienced a pterional approach ( n=31 ) , interhemispheric approach ( n=8 ) , or combined pterional and interhemispheric approach ( n=3 ) . meanwhile , the demographic and aneurysmal characteristics of the 376 patients who underwent surgical clipping ( n=188 ) or endovascular coiling ( n=188 ) for a single uia are summarized in table 1 . the ages , genders , and aneurysm locations were comparable between the surgical and endovascular groups . however , the diameters of the endovascularly - treated aneurysms were larger than those of the surgically - treated aneurysms , although the difference was small ( meansd , range : 5.92.8 mm , 2.2 - 16.0 mm vs. 5.12.2 mm , 2.0 - 14.0 mm ) when comparing the total hospital costs for surgical clipping of a single uia ( n=188 ) and endovascular coiling of a single uia ( n=188 ) , surgical treatment [ meansd : 8,280,0001,490,000 ( $ 75271355 ) ] showed significantly lower total hospital costs than endovascular treatment [ meansd : 11,700,0003,050,000 ( $ 106362773 ) , p<0.001 ] ( table 2 ) . the median and interquartile range ( iqr ) of the total hospital costs for surgical versus endovascular treatment were 7,770,000 ( $ 7064 ) and 1,440,000 ( $ 1309 ) vs. 11,440,000 ( $ 10400 ) and 3,460,000 ( $ 3145 ) , respectively . thus , the median of the total hospital costs for coiling was 47% higher than that for clipping . the higher total hospital costs for endovascular treatment were due to higher operative costs , including the medical equipment and endovascular coils . the operative costs for surgical clipping [ meansd : 5,800,000680,000 ( $ 5273618 ) ] were significantly lower than the operative costs for endovascular coiling [ meansd : 9,310,0002,800,000 ( $ 84642545 ) , p<0.001 ] . the median and iqr of the operative costs for surgical versus endovascular treatment were 5,600,000 ( $ 5091 ) and 730,000 ( $ 664 ) vs. 9,140,000 ( $ 8309 ) and 3,430,000 ( $ 3118 ) , respectively . the price of one endovascular coil was approximately 550,000 ( $ 500 ) , while the price of one surgical clip was approximately 140,000 ( $ 127 ) . in addition , endovascular treatment of a single aneurysm required a considerable increase in the number of coils according to the diameter of the aneurysm , whereas most surgical cases only required one to three aneurysm clips . notwithstanding , there was no statistical difference in the perioperative costs between the surgical and endovascular groups [ meansd : 2,480,0001,070,000 ( $ 2255973 ) vs. 2,400,0001,310,000 ( $ 21821191 ) ] . in the multi regression analysis , the factors significantly associated with the total hospital costs for endovascular treatment of a single uia were the aneurysm diameter ( p<0.001 ) and patient age ( p=0.014 ) , while the patient gender and aneurysm location were not associated . in the case of surgical treatment , the only factor significantly associated with the total hospital costs was the aneurysm diameter ( p<0.001 ) , while the patient age , gender , and aneurysm location were not associated . the pearson correlation analysis of the endovascular group showed a strong positive correlation ( r=0.77 ) between the aneurysm diameter and the total hospital costs ( fig . 1a ) , whereas only a weak positive correlation ( r=0.16 ) was revealed between the patient age and the total hospital costs . meanwhile , the pearson correlation analysis of the surgical group showed a weak positive correlation ( r=0.26 ) between the aneurysm diameter and the total hospital costs ( fig . a simple linear regression analysis was used to develop an equation for predicting the dependent variable ( y , total hospital costs ) given a value ( x , aneurysm diameter ) for the independent variable . for the endovascular group , this equation was y ( )= 6,658,630 + 855,250x ( mm ) or y ( $ ) = 6053.3 + 777.5x ( mm ) . the value of r was 0.6004 . thirty four patients were surgically treated for two ( n=27 ) or three ( n=7 ) uias during a single operation , however , no patient received endovascular treatment for multiple uias during a single session . the mean value of the total hospital costs for the surgical treatment of two uias was 10,810,0002,960,000 ( $ 98272691 ) , while the mean value for the surgical treatment of three uias was 11,540,0001,430,000 ( $ 104911300 ) . for both treatment groups , no direct mortality or permanent morbidity adversely affecting the glasgow outcome scale ensued from the treatments . two patients in the surgical group developed venous infarction and transient somnolence after clipping a pericallosal artery aneurysm , yet both patients recovered without neurological deficits . meanwhile , a patient in the endovascular group developed a thromboembolism and transient leg monoparesis after coiling an anterior communicating artery aneurysm . surgical clipping was applied to 222 patients , where a single aneurysm was clipped in 188 patients and 2 - 3 multiple aneurysms were clipped in 34 patients . while most patients underwent a superciliary keyhole approach ( n=180 , 81.1% ) , the others experienced a pterional approach ( n=31 ) , interhemispheric approach ( n=8 ) , or combined pterional and interhemispheric approach ( n=3 ) . meanwhile , the demographic and aneurysmal characteristics of the 376 patients who underwent surgical clipping ( n=188 ) or endovascular coiling ( n=188 ) for a single uia are summarized in table 1 . the ages , genders , and aneurysm locations were comparable between the surgical and endovascular groups . however , the diameters of the endovascularly - treated aneurysms were larger than those of the surgically - treated aneurysms , although the difference was small ( meansd , range : 5.92.8 mm , 2.2 - 16.0 mm vs. 5.12.2 mm , 2.0 - 14.0 mm ) when comparing the total hospital costs for surgical clipping of a single uia ( n=188 ) and endovascular coiling of a single uia ( n=188 ) , surgical treatment [ meansd : 8,280,0001,490,000 ( $ 75271355 ) ] showed significantly lower total hospital costs than endovascular treatment [ meansd : 11,700,0003,050,000 ( $ 106362773 ) , p<0.001 ] ( table 2 ) . the median and interquartile range ( iqr ) of the total hospital costs for surgical versus endovascular treatment were 7,770,000 ( $ 7064 ) and 1,440,000 ( $ 1309 ) vs. 11,440,000 ( $ 10400 ) and 3,460,000 ( $ 3145 ) , respectively . thus , the median of the total hospital costs for coiling was 47% higher than that for clipping . the higher total hospital costs for endovascular treatment were due to higher operative costs , including the medical equipment and endovascular coils . the operative costs for surgical clipping [ meansd : 5,800,000680,000 ( $ 5273618 ) ] were significantly lower than the operative costs for endovascular coiling [ meansd : 9,310,0002,800,000 ( $ 84642545 ) , p<0.001 ] . the median and iqr of the operative costs for surgical versus endovascular treatment were 5,600,000 ( $ 5091 ) and 730,000 ( $ 664 ) vs. 9,140,000 ( $ 8309 ) and 3,430,000 ( $ 3118 ) , respectively . the price of one endovascular coil was approximately 550,000 ( $ 500 ) , while the price of one surgical clip was approximately 140,000 ( $ 127 ) . in addition , endovascular treatment of a single aneurysm required a considerable increase in the number of coils according to the diameter of the aneurysm , whereas most surgical cases only required one to three aneurysm clips . notwithstanding , there was no statistical difference in the perioperative costs between the surgical and endovascular groups [ meansd : 2,480,0001,070,000 ( $ 2255973 ) vs. 2,400,0001,310,000 ( $ 21821191 ) ] . in the multi regression analysis , the factors significantly associated with the total hospital costs for endovascular treatment of a single uia were the aneurysm diameter ( p<0.001 ) and patient age ( p=0.014 ) , while the patient gender and aneurysm location were not associated . in the case of surgical treatment , the only factor significantly associated with the total hospital costs was the aneurysm diameter ( p<0.001 ) , while the patient age , gender , and aneurysm location were not associated . the pearson correlation analysis of the endovascular group showed a strong positive correlation ( r=0.77 ) between the aneurysm diameter and the total hospital costs ( fig . 1a ) , whereas only a weak positive correlation ( r=0.16 ) was revealed between the patient age and the total hospital costs . meanwhile , the pearson correlation analysis of the surgical group showed a weak positive correlation ( r=0.26 ) between the aneurysm diameter and the total hospital costs ( fig . a simple linear regression analysis was used to develop an equation for predicting the dependent variable ( y , total hospital costs ) given a value ( x , aneurysm diameter ) for the independent variable . for the endovascular group , this equation was y ( )= 6,658,630 + 855,250x ( mm ) or y ( $ ) = 6053.3 + 777.5x ( mm ) . thirty four patients were surgically treated for two ( n=27 ) or three ( n=7 ) uias during a single operation , however , no patient received endovascular treatment for multiple uias during a single session . the mean value of the total hospital costs for the surgical treatment of two uias was 10,810,0002,960,000 ( $ 98272691 ) , while the mean value for the surgical treatment of three uias was 11,540,0001,430,000 ( $ 104911300 ) . for both treatment groups , no direct mortality or permanent morbidity adversely affecting the glasgow outcome scale ensued from the treatments . two patients in the surgical group developed venous infarction and transient somnolence after clipping a pericallosal artery aneurysm , yet both patients recovered without neurological deficits . meanwhile , a patient in the endovascular group developed a thromboembolism and transient leg monoparesis after coiling an anterior communicating artery aneurysm . previous cost comparisons of surgical clipping and endovascular coiling of intracranial aneurysms have revealed distinct disparities between different countries . the international subarachnoid aneurysm trial ( isat ) , primarily conducted in europe , showed no significant cost difference between surgical and endovascular groups at a 1-year or 2-year follow - up13 ) . while the endovascular patients had higher costs related to the initial intervention and subsequent procedures due to recurrences , this was offset by lower costs related to the shorter hospital stay and fewer complications . however , a national analysis using a nationwide inpatient sample in the united states showed significantly higher total hospital costs for surgical clipping of both ruptured and unruptured aneurysms when compared with endovascular coiling3 ) . in contrast , the present study showed significantly lower costs for surgical clipping of uias , attributable to the lower device cost of clips over coils and good surgical results with minimal complications . in the present surgical case series , the advantage of lower economic costs was also enhanced by the reduced surgical invasiveness of a keyhole approach used on 81% of the patients . radovanovic et al.12 ) previously reported that minimally invasive surgery reduced hospital costs by decreasing the operative time and length of the hospital stay when compared with conventional surgery . however , in the present study , the impact of minimally invasive surgery on the treatment costs was not critical , as postoperative costs including hospital bed charges are not high in south korea . since the total hospital costs associated with endovascular coiling of a uia varied predictably as a function of the aneurysm diameter , the costs for endovascular coiling of a uia can be estimated before the procedure using the suggested equation provided by a simple linear regression analysis , where r is a statistical measure of the closeness of the data to the fitted regression line . thus , when the value of r is 0.6 , 60% of the data is on the straight line . first , it is based on a retrospective review of a case series from a single institution . as the surgical techniques and results affecting treatment costs can vary significantly between medical institutions , it is difficult to make generalizations . second , it does not include any long - term economic impact related to the surgical and endovascular treatment of uias . for example , the number of postoperative angiograms , outpatient services , and retreatments can be higher in the case of endovascular treatment . in south korea , the surgical clipping of uias was found to incur lower total hospital costs than endovascular coiling when the surgical results were favorable without significant complications . plus , a strong positive correlation was noted between an increase in the aneurysm diameter and a dramatic increase in the costs of endovascular coiling .

we describe a mass spectrometry method , quantmode , which improves the accuracy of isobaric tag based quantification by alleviating the pervasive problem of precursor interference co - isolation of impurities through gas - phase purification . quantmode analysis of a yeast sample contaminated with interfering human peptides showed substantially improved quantitative accuracy compared to a standard scan , with a small loss of spectral identifications . this technique will allow large - scale , multiplexed quantitative proteomics analyses using isobaric tagging . objectivea cost comparison of the surgical clipping and endovascular coiling of unruptured intracranial aneurysms ( uias ) , and the identification of the principal cost determinants of these treatments.methodsthis study conducted a retrospective review of data from a series of patients who underwent surgical clipping or endovascular coiling of uias between january 2011 and may 2014 . the medical records , radiological data , and hospital cost data were all examined.resultswhen comparing the total hospital costs for surgical clipping of a single uia ( n=188 ) and endovascular coiling of a single uia ( n=188 ) , surgical treatment [ meanstandard deviation ( sd ) : 8,280,0001,490,000 ] resulted in significantly lower total hospital costs than endovascular treatment ( meansd : 11,700,0003,050,000 , p<0.001 ) . in a multi regression analysis , the factors significantly associated with the total hospital costs for endovascular treatment were the aneurysm diameter ( p<0.001 ) and patient age ( p=0.014 ) . for the endovascular group , a pearson correlation analysis revealed a strong positive correlation ( r=0.77 ) between the aneurysm diameter and the total hospital costs , while a simple linear regression provided the equation , y ( )=6,658,630 + 855,250x ( mm ) , where y represents the total hospital costs and x is the aneurysm diameter.conclusionin south korea , the total hospital costs for the surgical clipping of uias were found to be lower than those for endovascular coiling when the surgical results were favorable without significant complications . plus , a strong positive correlation was noted between an increase in the aneurysm diameter and a dramatic increase in the costs of endovascular coiling .

intensive care requires not only the use of sophisticated equipment but also highly skilled and dedicated healthcare staff . as such , the intensive care unit ( icu ) takes a significant proportion of the total healthcare cost , and therefore patients with prolonged icu length of stay ( los ) can have serious cost implications . moreover , patients with prolonged icu - los can also lead to a shortage of icu beds and result in operations being cancelled . regarding the population of cardiac surgery patients , the prediction of their icu - los is a fact of great significance , based on the need for containing the burgeoning costs involved in cardiac operations , saving resources and icu costs . in addition , the cardiac surgery icu - los is a significant healthcare index that has been correlated with poor patient outcome , such as increased expenditure and higher morbidity and mortality rates . furthermore , the icu - los is a valuable indicator of the quality and the effectiveness of the provided care . the above data could interpret the scientific interest for the early prediction of the length of hospitalization in cardiac surgery icus . various predictive models for prolonged icu stay after cardiac surgery have been developed , during the last two decades [ 5 , 6 ] , and several papers have attended preoperative risk factors associated with prolonged icustay . these efforts are now more than ever in season and necessary because of the global financial crisis , that has affected negatively the healthcare systems ' operation , worldwide , and demands a more efficient use of the icu resources . the aim of this study was to identify the preoperative and intraoperative predictors for prolonged icu - los , among cardiac surgery patients . this identification could lead to the early recognition of the high - risk patients for longer icu hospitalization and , also , could provide an important aid to nursing administrators and leaders for the appropriate resource planning and effective choice of the operation list , with the low - risk patients being scheduled for surgery before the high - risk candidates . we conducted an observational cohort study from september 2010 to january 2011 among 194 patients who were admitted to the 8-bed cardiac surgery icu of a general , tertiary hospital of athens , greece . the inclusion criteria into the study were a priori defined as follows : ( a ) patient age 18 years old and ( b ) a minimum icu - los of 24 hours . based on the predefined inclusion criteria 44 patients were excluded from the study , and 150 met the inclusion criteria and constituted our study sample . a short structured questionnaire on basic sociodemographic and clinical patient characteristics was used for data collection purposes . the collected sociodemographic data included patient age , gender , height , weight , and body mass index ( bmi ) . in addition , the collected clinical characteristics were the history of chronic pulmonary disease and diabetes , the type of surgery , the preoperative serum creatinine ( cr ) levels , the blood glucose levels during the intraoperative period , the use of intraaortic balloon pump ( iabp ) preoperatively , the application of cardiopulmonary bypass ( cpb ) , the preoperative ejection fraction of the left ventricle ( eflv ) , the duration of surgery and cpb , the presence of atrial fibrillation ( af ) preoperatively , the emergency procedure , the ischemic time , the transfusion of red blood cells ( rbcs ) , the euroscore values and the icu - los . we defined the icu - los as the duration of the hospitalization of patients from their admission to the icu until their discharge . euroscore is the most valid and reliable stratification model for predicting the perioperative risk of cardiac surgery patients in north america , europe , and japan . it was developed between 1995 and 1999 and was first introduced into clinical practice in 1999 , while the logistic algorithm of euroscore is available since 2003 . it includes three wide categories of risk factors : the patient- ( age , female sex , chronic pulmonary disease , extracardiac arteriopathy , neurological dysfunction disease , previous cardiac surgery , serum creatinine preoperatively , active endocarditis , critical preoperative state ) , the cardiac- ( unstable angina , left ventricular dysfunction moderate : left ventricular ejection fraction ( lvef ) 3050% or poor : lvef < 30% , recent myocardial infarct ( < 90 days ) , pulmonary hypertension , and the operation- ( emergency , other than isolated coronary artery bypass grafting , surgery on thoracic aorta , postinfarct septal rupture ) associated factors . the sum of the various risk factors results in a total score which represents the predicted probability of mortality and ranges between 0 and 100% ( logistic euroscore ) [ 8 , 9 ] . one of the researchers , the same each time , obtained the data based on the review of both medical and nursing patient records . euroscore was calculated for each patient based on data collected by the hospital records . in total finally , the measurement of the serum glucose levels was carried out for each patient , intraoperatively , immediately after the induction of anesthesia and was repeated every 30 minutes . continuous variables are presented as median ( interquartile range , ir ) , while categorical variables are presented as absolute and relative frequencies . the kolmogorov - smirnov test and graphs ( histograms and normal q - q plots ) were used to test the normality of the distribution of the continuous variables . distributions of continuous variables were extremely skewed and were converted into binary variables using median values as the criterion for separation . for bmi the criterion for separation was the international limit of 24.9 kg / m . for serum cr levels the cut - off point was the 1.4 mg / dl , a value that constitutes the limit between the normal and the increased cr levels of the biochemistry laboratory of the hospital in which our study was carried out . in addition , the cut - off for prolonged icu - los ( 2 days ) was chosen based on the median value and the literature data [ 3 , 10 ] . chi - square test and fisher 's exact test were used to identify differences between groups . kendall 's tau rank correlation coefficient ( r ) was used to estimate correlation between continuous variables . variables that were statistically significant ( p < 0.05 ) in bivariate analysis were entered into the backward stepwise multivariate logistic regression analysis with icu - los as dependent variable . multivariate analysis was used to control potential confounding variables . only variables with 95% confidence interval for adjusted odds ratio that did not cross 1 were considered to have an independent and significant association with icu - los . criteria for entry and removal of variables were based on the likelihood ratio test , with entrance and removal limits set at p < 0.05 and p > 0.05 . we estimated crude and adjusted odds ratios ( ors ) with 95% confidence intervals ( cis ) for the predictive factors related to the icu - los . a correlation matrix was assessed prior to conducting the multivariate logistic regression analysis to check for collinearity among the independent variables . kendall 's tau rank correlation coefficient ( r ) was used to estimate correlations between variables . all tests of statistical significance were two - tailed , and p - values of less than 0.05 were considered significant . statistical analysis was performed using the statistical package for social sciences software ( ibm spss statistics 19.0 for windows , spss inc . , researchers received patients ' consent after being informed about the type and purpose of the study . each potential subject adequately informed of the aims , method , the anticipated benefits , and potential risk of the study . the potential subject informed of the right to refuse to participate in the study or to withdraw consent to participate any time without reprisal . the investigation was carried out in accordance with the ethical standards of the responsible institutional committee for human experimentation and with the helsinki declaration of 1975 , as revised in 2008 . precautions took place to protect the privacy of research subjects and the confidentiality of their personal information . the methods of the study were restricted to observation and recording patient data and no part of the standard care was omitted . the majority of patients were males ( 68% ) , and the median age was 66 ( ir 15 ) years old . most patients had either received a coronary artery bypass grafting ( 44.7% ) or had undergone a valve disease surgery repair or replacement ( 27.3% ) . 17.3% and 48% of patients had serum cr levels higher than 1.4 mg / dl and blood glucose levels higher than 130 mg / dl , respectively . preoperative af was presented in 11.7% of patients , while the median logistic euroscore value was 5.4 ( 7.3 ) % . the median icu - los was 2 ( ir 3 ) days , and the overall 30-day mortality was 13.3% . table 2 provides the main predictive factors ' ( including demographic characteristics and perioperative - related factors ) distribution and their relation with icu - los ( below or above median ) . the preoperative serum cr > 1.4 mg / dl , the mean intraoperative blood glucose levels > 130 mg / dl , the duration of surgery and the duration of cpb longer than 250 and 125 min , respectively , the presence of af preoperatively , the emergency procedure , the transfusion of rbc and the logistic euroscore values > 5.4% were associated with icu - los more than 2 days at the level of 5% ( p a correlation matrix was assessed prior to conducting the multivariate logistic regression analysis to check for collinearity among the independent variables . because duration of surgery and duration of cardiopulmonary bypass were strongly correlated ( r = 0.66 , p < 0.001 ) , we chose to include only the duration of surgery in the multivariate model . we found a positive association between perioperative risk ( high euroscore ) and icu - los ( or = 4.3 , 95% ci = 2.28.4 , p < 0.001 ) while this association remained statistically significant after adjusting for gender , age , bmi , chronic pulmonary disease , diabetes , type of surgery , preoperative iabp , cbp , eflv , duration of surgery , cardiopulmonary bypass time , emergency , ischemic period , and rbc ( or = 2.6 , 95% ci = 1.25.6 , p = 0.017 ) . in addition to the above , patients with preoperative serum cr > 1.4 mg / dl had an almost 3 times greater probability to have icu - los > 2 days ( or = 3 , 95% ci = 18.7 , p = 0.049 ) , and also patients with mean intraoperative blood glucose > 130 mg / dl had an almost 3 times greater probability for longer icu - los ( or = 3 , 95% ci = 1.46.4 , p = 0.004 ) . finally , patients with af preoperatively were associated with an almost 6.3 times greater probability of having an icu - los of more than 2 days . the main findings of our study were the strong association of three preoperative ( serum cr levels , preoperative af , and logistic euroscore ) and one intraoperative factor ( glucose levels ) with the prolonged icu - los of cardiac surgery patients . in particular , patients with preoperative renal dysfunction , high perioperative risk , history of af or high mean glucose levels intraoperatively have greater possibility to stay in the cardiac surgery icu more than 2 days . as aforementioned , the increased serum cr levels , preoperatively , were statistically significantly correlated with prolonged icu hospitalization . in line with our findings , herman et al . , in a recent study of 3489 cabg patients in a hospital of canada , concluded that the history of renal failure is among the independent predictors for prolonged hospitalization into the intensive care setting . in addition , ranucci et al . , in a retrospective study of 9120 cardiac surgery patients revealed the increased preoperative cr levels among the main determinants of late discharge from the icu . the history of renal failure was among the independent risk factors for extended cardiac surgery icu hospitalization . similar conclusions were reached by the studies of bucerius et al . and ghotkar et al . . however , in a recent retrospective study of 298 patients who underwent aortic arch surgery , the authors found that the increased serum cr levels were associated with prolonged icu stay , through the univariate analysis . nevertheless , contrary to the finding of this study , the multivariate analysis did not indicate significant association between these two variables . in addition , wong et al . correlated the high cr levels postoperatively and not preoperatively with increased cardiac surgery icu hospitalization . we could find easily a logical interpretation regarding the previous correlation , based on the fact that patients with preexisting renal dysfunction or / and failure are characterised as high - risk patients with higher clinical severity and morbidity rates . in addition , these patients have higher incidence of postoperative complications , such as hemorrhage , reoperation , prolonged mechanical ventilation , strokes , and necessity for renal replacement therapy [ 1517 ] . moreover , the literature review reveals that the renal dysfunction during the preoperative period , among cardiac surgery patients , is a strong and independent indicator of increased in - hospital and 30-day mortality . consequently , their stay for a longer period in the icu in order to achieve clinical stability and to discharge from the critical care environment safely is rather reasonable . another important finding of this study was the significant correlation between the preoperative af and the prolonged icu - los . , in a prospective study of 10759 patients undergoing cabg , observed that 20 variables ( 15 preoperative and 5 intraoperative ) , including the presence of af preoperatively , have a strong association with prolonged icu - los . moreover , the authors of other studies [ 19 , 20 ] found that the no - sinus rhythm or the rhythm disturbances , preoperatively , including af , are significant predictors of prolonged stay in the cardiac surgery icu . the recent developments in invasive cardiology , the improvement of surgical techniques , and the improved pharmacological treatments have contributed to the application of surgical treatment among elder patients with advanced cardiovascular disease and significant comorbidity [ 13 , 21 ] . therefore , af appears very frequently preoperatively in patients who are candidates for cardiac surgery , increasing the perioperative risk and the occurrence of postoperative complications . preoperative af significantly worsens the heart functional status , increasing the incidence of postoperative complications , such as delirium , stroke , and low cardiac output syndrome , which lead to negative healthcare patient outcomes , including higher mortality and prolonged icu and in - hospital stay [ 2123 ] . in addition , a point of great interest is that while almost 30% of patients undergoing cardiac surgery will develop postoperative af , if patients have history of af ( preoperative af ) , this probability becomes almost 2 times greater ( 60% ) [ 24 , 25 ] . the above data in conjunction with the correlation between the postoperative af and the prolonged cardiac surgery icu - los allow us to interpret the association of the preoperative af with the prolonged icu hospitalization , a finding which was revealed by our study . in addition , via the multivariate analysis , we found that patients with high euroscore have significantly greater probability to stay longer in the icu than patients with lower perioperative risk . , in a recent study of 313 consecutive cardiac surgery patients , concluded that there is a strong association between high perioperative risk and the prolonged icu - los . in particular , patients with high logistic euroscore values had an almost 1.9 times greater probability to stay in the icu longer than 2 days . several studies have reached to similar findings , revealing a positive association between increased perioperative risk ( high euroscore values ) and the increased cardiac surgery icu - los [ 10 , 26 , 27 ] . in addition , several investigators using risk stratification models other than euroscore , such as parsonnet score , noted , in line with our findings , strong evidence between the high perioperative risk and the increased icu - los [ 6 , 12 , 28 ] . the above correlation could be justified by the fact that variables , which are included in euroscore , constitute significant risk factors and predictors of the increased hospitalization in the critical care environment . older age , female gender , chronic pulmonary disease , extracardiac arteriopathy , neurological dysfunction , high preoperative cr levels , previous cardiac surgery , unstable angina , poor left ventricular ejection fraction , recent myocardial infarction , emergency operation and procedures other than isolated coronary artery bypass grafting are parameters which are included in euroscore model and simultaneously have been independently and separately correlated with increased icu - los . indeed , a recent systematic review and validation study of the performance of 14 icu - los prediction models among cardiac surgery patients revealed that the euroscore model has significantly high performance in terms of discrimination , accuracy , and calibration . among the 14 prediction models , these data indicate that euroscore is a valid and reliable tool for predicting the cardiac surgery icu - los . moreover , we identified the significant correlation of one intraoperative variable with the prolonged icu - los . in particular , patients with mean serum glucose levels > 130 mg / dl , intraoperatively , had greater probability to stay longer in the icu . in accordance with our results , ouattara et al . in a prospective cohort study of 200 diabetic patients undergoing coronary artery bypass grafting concluded that the poor glycemic control intraoperatively is correlated with significant increase in the icu - los . hyperglycemia during the perioperative period has been associated with negative patient outcome and constitutes an independent risk factor for higher morbidity and mortality . moreover , the poor glycemic control has been associated with postoperative complications , such as sternal wound infection , impaired wound healing , adverse renal and pulmonary events , increased incidence of af and myocardial infarction [ 29 , 3135 ] . however , in contrary to the findings of the present study , the literature review shows that many investigators have not achieved to correlate the intraoperative hyperglycemia with the prolonged icu - los among cardiac surgery patients [ 3538 ] . in addition , lazar et al . in a controlled randomized trial of 141 diabetics patients who underwent coronary artery bypass graft surgery correlated the inadequate intra- and postoperative glycemic control with increased total postoperative in - hospital stay and not icu stay separately . the significant shortage of randomized controlled trials and the different definitions of the perioperative hyperglycemia in conjunction with the different intensity and duration of the applied serum glucose therapy do not allow safe conclusions and could justify the inconsistence of the literature findings [ 39 , 40 ] . finally , the median icu - los of our study was 2 days , a finding that is in line with the international literature , based on the fact that most researchers have demonstrated the 2 days as the cutoff point for the increased cardiac surgery icu - los [ 3 , 10 ] . our study allows us to reach significant conclusions regarding the preoperative and intraoperative factors that could influence the icu - los among cardiac surgery patients . in addition , to the best of our knowledge , this study is among the few greek studies aiming to investigate parameters that could predict the length of icu stay and consequently to contribute to administrative decisions regarding the plan of the operative list , the provided care , and the icu resources ' allocation . however , our study has some limitations . firstly , the small sample size in conjunction with the fact that this study was carried out in one cardiac surgery centre limits the generalization of the finding to the general cardiac surgery population and affects the external validity of the study . secondly , the icu - los is often a subjective outcome that affected , except from clinical ( e.g. , patient severity , complications ) and by nonclinical factors , such as the icu and the ward capacity of the institution , the opinion of the intensive care specialists or a busy operation schedule . moreover some hospitals are more comfortable transferring patients out to a lower acuity nursing environment than other hospitals . finally , the different cut - off points of the prolonged icu - los impede the ability to compare the findings of the corresponding international literature . further research is needed to identify more predictive variables for the identification of patients at risk for prolonged cardiac surgery icu hospitalization . demographic and clinical preoperative and intraoperative patient characteristics are significant predictors of the icu - los among cardiac surgery patients . patients with renal dysfunction , history of af , high euroscore values , and intraoperative hyperglycemia seem to have significantly greater probability of staying for more than 2 days in the intensive care environment . the early recognition of the nonmodified parameters ( history of renal dysfunction and af , high euroscore ) could lead to the early identification of patients at risk . in addition , the management of the modified predictor ( intraoperative hyperglycemia ) , through the maintenance of normoglycemia during the operation , could erase a significant risk factor for prolonged icu - los in the cardiac surgery setting . nursing administrators may use this knowledge for the appropriate use of the limited icu , human and financial , resources . in addition , they have the opportunity for a more efficient and effective planning of the operative list , selecting low - risk patients in periods with significant restriction of the icu beds availability . this chance has great importance for healthcare systems , such as the greek , that are characterized by limited resources , nursing staff and icu - beds shortage , mainly within the public sector . colorectal cancer ( crc ) is one of the most prevalent cancer , along with lung and breast cancers . in 2012 , approximately 1.4 million new cases and 700,000 crc - related deaths were estimated worldwide ( 1 ) . over the last decade , crc incidence and mortality in the united states have been on a steady decline as a result of advances in its prevention , screening , and treatment . nevertheless , it is still the second leading cause of cancer deaths ( 2 ) . moreover , there has been a significant rise in crc incidence in newly developed or economically transitioning countries , such as south america , eastern european countries , and most parts of asia ( 3 ) . although crc is highly curable at an early stage , approximately 50% of patients with crcs eventually develop metastatic disease ( 4 ) . five - year survival rate is approximately 10% in patients with a stage iv disease ( 5 ) . there are many known risk factors for crc including age , sex , smoking , excessive alcohol consumption , high intake of red and processed meat , obesity , diabetes , inflammatory bowel disease , inherited genetic disorder , and family history of crc ( 6789101112 ) . among them , ulcerative colitis and crohn 's disease increase the overall risk of colitis - associated cancer ( 14 ) . approximately 20% of patients with inflammatory bowel disease develop colitis - associated cancer within 30 years of disease onset ( 15 ) . inherited crc , including lynch syndrome and familial adenomatous polyposis , represent less than 5% of crc cases ( 16 ) . in this review , we briefly review currently available drugs for the treatment of colorectal cancer and summarize clinical studies that use cell - based cancer immunotherapy as promising therapeutic strategy . we also suggest the use of cytokine - induced killer ( cik ) cells as an additional therapeutic approach for metastatic colorectal cancer . most crcs occur without a known genetic risk factor and significant family history . only a small proportion ( 5% ) of inherited crcs , which is also known as familial crc , have been thoroughly characterized ( 17 ) , and additional 20~25% are yet to be completely understood ( 17 ) . crc develops over the course of more than 10 years , and entails multistep genetic events for tumor progression . a multistep genetic model of colorectal carcinogenesis colorectal cancers occur as a result of oncogenes activation , along with the inactivation of tumor suppressor genes by mutations or loss of heterozygosity ( loh ) ( 18 ) . mutations in at least four to five genes are required for the formation of a malignant tumor ( 18 ) . the molecular pathogenesis of colorectal cancer is commonly classified into two main pathways of genomic instability : chromosomal instability ( cin ) and microsatellite instability ( msi ) pathways . cin leads to an imbalance in chromosome number ( aneuploidy ) such as chromosome gains or losses ( including loh events at specific tumor suppressor gene loci ) , and is observed in 80~85% of sporadic crcs ( 18192021 ) . a mutation coupled with loh of the adenomatous polyposis coli ( apc ) tumor suppressor gene , followed by activating mutations of v - ki - ras2 kirsten rat sarcoma viral oncogene homolog ( kras ) , lead to the initial appearance of adenoma from normal colonic epithelium ( 18 ) . apc mutations are typically observed in earliest stages of tumors , including in adenomas as small as 0.5 cm ( 22 ) . this indicates that a functional loss of apc , which is part of the wnt signaling pathway , plays a role in early tumorigenesis . in contrast , msi , which is observed in 15~20% of sporadic crcs ( 21 ) , is a hypermutable phenotype caused by impaired dna mismatch repair ( mmr ) system to correct errors such as single - base mismatches and insertion - deletion loops that spontaneously occur during dna replication ( 2324 ) . msi arises from impaired dna mmr genes , such as mutl homolog 1 ( mlh ) 1 , muts homolog ( msh ) 2 , and to a lesser degree msh6 ( 2526 ) . faulty dna fidelity caused by defects in dna mmr causes frameshift and point mutations , mainly in repetitive sequences ( microsatellites ) ( 26 ) . lynch syndrome is a well - established hereditary predisposition to colorectal cancer caused by a germline mutation in a dna mmr gene . however , 12% of msi exhibit sporadic crc with cin ( 27 ) . because pathways are not mutually exclusive , crc can display features of multiple pathways . i colon cancer , surgery is the definitive treatment without the use of adjuvant chemotherapy . benefit of adjuvant chemotherapy for patients with a stage ii disease is highly debatable due to minimal gains in overall response . however , adjuvant chemotherapy is generally acceptable for the use patients with node - positive cancer following surgery ( high - risk stage ii disease ) ( 29 ) . adjuvant chemotherapy is required for all patients with a stage iii colon cancer after surgical resection , which have a high risk of recurrence of 15~50% ( 30 ) . conventional chemotherapeutic agents include 5-fluorouracil ( 5-fu ) with leucovorin ( lv ) , capecitabine ( an orally administered prodrug of 5-fu ) , oxaliplatin , and irinotecan . adjuvant chemotherapy using the 5-fu / lv regimens following surgery provides significant reduction in mortality by 22% and improves event - free survival by 35% ( 31 ) . to improve disease - free survival ( dfs ) and overall survival ( os ) , currently , folfox ( 5-fu / lv and oxaliplatin ) or folfiri ( 5-fu / lv and irinotecan ) is widely used for standard first- or second - line treatment in patients with node - positive colon cancer following surgical resection ( 3233 ) . however , folfox and folfiri demonstrated greater toxicities , including grade 3 or higher acute toxicity , compared to 5-fu / lv alone ( 34 ) . in 2004 , targeted therapeutic agents for metastatic colorectal cancer , such as anti - epidermal growth factor receptor ( egfr ) antibodies ( cetuximab and panitumumab ) , vascular endothelial growth factor ( vegf ) inhibitors ( bevacizumab , ziv - aflibercept , and ramucirumab ) , and a multikinase inhibitor ( regorafenib ) , were approved by the us food and drug administration ( fda ) for combinational use with standard chemotherapy treatment ( 35 ) . currently , there are various regimens of combinational therapies using targeted therapeutic agents and conventional chemotherapy drugs ( 36 ) . treatment strategy is often dependent on tumor - specific factors , such as kras and/or v - raf murine sarcoma viral oncogene homolog b ( braf ) mutation , and condition of patients and their disease progression following first - line treatment ( 3336 ) . since ras mutations lead to continuous activation of signaling pathways downstream of egfr , the use of anti - egfr antibodies is effective in patients with wild - type kras exon 2 , but not in patients with kras and/or braf mutations . thus , approximately 30~40% of crcs are associated with kras gene mutation and do not respond to anti - egfr antibodies ( 37 ) . over the past decade , multidisciplinary advances in the treatment of metastatic colorectal cancer with targeted therapy have statistically improved progression - free and overall survivals . despite these advances , only a relatively small effect on survival outcomes was observed , especially in patients with distant metastatic disease . dendritic cell ( dc ) therapy is one of several strategies in therapeutic cancer vaccines . sipuleucel - t , a dc - based cancer vaccine , has been approved by the us fda for advanced prostate cancer in 2011 . the goal of dc vaccination is to elicit anti - tumor response by inducing tumor - specific effector t cells . dcs are generated ex vivo by culturing hematopoietic progenitor cells or monocytes with a combination of cytokines and then pulsing them with antigens ex vivo ( 38 ) . there are various type of cancer antigens for dcs loading , including mutated and non - mutated antigens ( 39 ) . now , we can find 9 clinical trials for the treatment of patients with crc from 2004 to 2015 ( table 1 ) . antigens that are most commonly used for colorectal cancer include carcinoembryonic antigen ( cea ) peptides ( 4041 ) , melanoma - associated antigen ( mage ) from allogeneic melanoma cell lysates ( 4243 ) , and autologous cell lysates from biopsy material ( 44 ) . while cea expression in normal colon epithelial cells is relatively low , it is over expressed in most colorectal carcinomas as well as in many cancers ( 45 ) . therefore , cea has been the major immunological target of dc - based cancer vaccines for colorectal cancer . clinical trials of cea - pulsed dcs demonstrated its immune - stimulatory capacity and it was well tolerated in patients without any observable toxicities . however , the overall clinical response was rather unimpressive ( 404146 ) , which may reflect severely impaired immune functions in patients with excessive tumor burdens and tumor immunoediting mechanisms . nevertheless , several clinical studies of mage - pulsed dcs showed 24~40% clinical benefit rate with durable responses and tumor regression ( 4243 ) . moreover , regulatory t cell levels declined upon dc vaccination ( 42 ) . recently , hunyadi j. and colleagues demonstrated that autologous tumor cell lysates - loaded dcs led to an increase in 6-years survival rate in colorectal cancer patients and more efficient induction of t - lymphocytes proliferation in vitro when compared to cea - pulsed dcs ( 44 ) . however , because of limited number of patients , additional evaluations in large - scale clinical trials are needed . although significant advances have been made over the past decade , further studies are required to fully determine the potential antitumor effects of dc vaccination for colorectal cancer . adoptive cell therapy ( act ) for metastatic melanoma was first described in 1988 ( 47 ) . in act , tumor - infiltrating lymphocytes ( tils ) are collected from solid tumor specimen , and are activated and expanded ex vivo . subsequently , tils are administered intravenously to the autologous patient . in 2002 , dudley me et al . demonstrated that host immunodepletion using cyclophosphamide and fludarabine prior to til infusion resulted in dramatic improvement in clinical outcomes for patients with metastatic melanoma ( 48 ) . although act has been evaluated in various cancers , only a small number of act trials for crc have been performed . in a phase i clinical trial , 14 patients with colorectal liver metastases received act with tils in combination with high - dose il-2 ( 49 ) . unfortunately , no significant difference in dfs rates was observed between tils therapy and conventional chemotherapy . a novel act approach for crc was conducted in two separate studies ; sentinel lymph node ( sln ) t lymphocytes were used in a pilot study ( seven with stage ii - iii and nine with stage iv crc ) ( 50 ) and in a phase i / ii study ( 46 with stage i - iii and nine with stage iv crc ) ( 51 ) , respectively . all patients with stage iv disease responded to the treatment with the following outcomes : four stable disease , one partial response , and four complete response with no detectable cancer cells remaining . the overall survival of act - treated patients was also significantly improved to 2.6 years compared to 0.8 years in conventionally - treated control patients . the latest phase i / ii study also showed favorable clinical outcomes ( 51 ) . the median overall survival of sln - t lymphocytes - treated and control groups in stage iv patients were 28 and 14 months , respectively . in the two studies , no significant toxicity was observed following sln - t lymphocytes treatment . these studies demonstrated that sln - t lymphocyte immunotherapy is indeed feasible and safe for patients with metastatic crc . however , surgeon 's proficiency may be required for the intraoperative sentinel node detection . in a phase i study of autologous genetically engineered t cells , all patients developed severe colitis despite showing a decrease in serum cea levels by cea - specific t cells ( 52 ) . cytokine - induced killer ( cik ) cells are ex vivo - expanded lymphocytes used for cancer immunotherapy . cik cells are generated by culturing peripheral blood mononuclear cells ( pbmcs ) with a combination of il-2 and anti - cd3 monoclonal antibodies for 14 days ( 53 ) . cik cells consist of heterogeneous cell population , mainly cd3cd56 , cd3cd56 , and a minor population of cd3cd56 cells . importantly , cd3cd56 cells are the distinguishing cell population among the cik cells and have the most potent cytotoxic function ( 53 ) . cd3cd56 cells originated from cd3cd8cd56 cells but not from cd3cd56 cells during the ex vivo expansion ( 54 ) . cik cell cytotoxicity is mediated by perforin release and dependent on several activating receptors such as nkg2d , nkp30 , and dnam-1 ( 5556 ) . cik cells also exhibit non - specific and non - mhc - restricted cytotoxicity ( 56 ) . over the past decade , cik cell therapy has been evaluated in numerous clinical studies in patients with various types of cancer , such as hepatocellular carcinoma , non - small cell lung cancer , renal cell carcinoma , and gastric cancer ( 57 ) . cik cell therapy can be used as a postoperative adjuvant treatment as well as a palliative treatment following standard therapies . cik cell therapy was evaluated in a limited number of clinical studies in patients with colorectal cancer . in a retrospective study , 21 of 96 colorectal cancer patients who underwent surgery as well as adjuvant chemotherapy received one to three cycles of cik cell transfusion for immunotherapy ( 58 ) . patients in the cik - treated group had significant improvement in their 2-year dfs rates than those in the control group ( 59.6524.80% vs 29.356.39% ) . other studies used cik in a combination therapy with dcs , specifically tumor lysate - pulsed dcs . in 2014 , two clinical studies of dc vaccine and cik cell combinational therapy for colorectal cancer patients were published . one study demonstrated that overall survival rates were significantly improved in the dc - cik therapy compared to the control group ( p=0.04 ) ( 59 ) . moreover , most patients that received dc - cik therapy displayed improvement in quality of life , including physical strength , appetite , sleep , and body weight . gao , d. and colleagues evaluated the clinical benefits of dc - cik therapy in 54 gastric and colorectal patients following surgery with or without chemo - radiotherapy ( 60 ) . the study demonstrated that dfs and 5-year survival of colorectal cancer patients were significantly prolonged in dc - cik treatment groups ( dfs rate : 66% and 5-year survival rates : 75% ) when compared to patients in control groups ( dfs rate : 8% and 5-year survival rates : 15% ; p<0.01 ) . il-12 levels were significantly increased in patients that received the dc - cik treatment . besides fever , since they did not compare the efficacy of combination therapy with each alone , we could not clarify whether combination therapy is better than each therapy . however , when we considered the efficacy of the single therapy of dcs and cik cells described above , we could presume that the combination of cik and dc might exert better antitumor activity than each alone . here , we provide additional evidence that cik cell therapy can effectively prevent growth of colorectal cancers in a xenograft mouse model . hypaque density gradient centrifugation , washed three times with pbs , and cultured in the presence of immobilized anti - cd3 antibody ( 5 g / ml ) and recombinant human il-2 ( 700 u / ml ) for five days . the cell suspension was further incubated in complete medium containing il-2 only ( 170 u / ml ) for nine days . thereafter , medium containing il-2 was replenished every 2 or 3 days . on day 14 cik cells comprised of 94% cd3 , 3% cd3cd56 , 16% cd3cd56 , 14% cd4 , and 81% cd8 cells ( fig . 1a ) . to examine the cytotoxicity of cik cells , two cell lines , sw620 and k-562 were used . sw620 cells are metastatic human colon cancer cell lines derived from lymph node ( 61 ) . k-562 cell lines are human leukemic cells commonly used as reference target cells of nk cells due to the very low level of mhc class i antigens on their cell membrane ( 62 ) . a 4-h cr release assay revealed that cik cells were able to kill 14% , 22% , and 36% of sw620 cells at effector : target ratios of 10:1 , 30:1 , and 100:1 , respectively ( fig . moreover , cik cells showed strong cytotoxicity against k562 cells , a known nk - sensitive target . a nude mouse xenograft assay was used to examine the in vivo antitumor activity of cik cells . sw620 cells ( 610 ) in 300 l of pbs were injected subcutaneously on day 0 , followed by intravenous once - a - week injection of cik cells and adriamycin ( adr ) . on day 25 , mice were sacrificed and the tumor mass and body weight ( to determine toxicity ) were measured . in control mice , sw620 cells grew to a tumor volume of 473190 mm ( n=7 ) ( fig . cik cells injected at doses of 110 , 310 , and 1010 cells per mouse were able to inhibit tumor growth by 7% , 53% , and 73% , respectively . cik cells injected at doses of 110 , 310 and 1010 cells per mouse reduced tumor weight by 10% , 54% and 73% , respectively ( fig . thus , our preclinical efficacy data showed that cik cells were able to kill sw620 cells in vitro and in vivo , suggesting that cik cells might be a good immunotherapy candidate for colorectal cancer . patients with metastatic colorectal cancer have significant risk of recurrence following surgery and conventional adjuvant therapy . although advances in therapeutic agents for crc have reduced the risk of recurrence and increased overall survival , patients with metastatic disease have a poor prognosis and a particularly low 5-year survival rates of less than 10% . additionally , current combination therapy is occasionally discontinued due to a grade 3~4 toxicity with negative effects on quality of life ( 63 ) . overall quality of life should be considered for patients with stage iv disease for palliative therapy . also , elderly patients with stage iii disease often avoid adjuvant chemotherapy due to anxiety of old age , comorbidities , side effects , and perceived minimal benefit ( 64 ) . clinical and preclinical studies of crc treatment with dcs , t cells , and cik cells showed promising outcomes , although only limited information have been available till recently . among the immune effector cells , cik cells have certain advantages in a clinical application : they are relatively easy to generate and expanded in large - scale from pbmcs , their non - mhc - restricted cytotoxic activity could eliminate mhc class i - negative tumor cells , and importantly , cik cell therapy causes mild , transient , and easy - to - manage side effects . over the last decades , cik cell therapy has been evaluated in numerous clinical studies in patients with various cancers . in most patients , combination therapy using conventional agents and cik cells showed superior clinical outcomes than standard therapy alone . clinical studies previously reported by others and our preclinical data suggest that immunotherapy of crc with cik cells can be a promising strategy to limit the growth and metastasis of crc .

the prediction of intensive care unit length of stay ( icu - los ) could contribute to more efficient icu resources ' allocation and better planning of care among cardiac surgery patients . the aim of this study was to identify the preoperative and intraoperative predictors for prolonged cardiac surgery icu - los . an observational cohort study was conducted among 150 consecutive patients , who were admitted to the cardiac surgery icu of a tertiary hospital of athens , greece from september 2010 to january 2011 . multivariate regression analysis revealed that patients with increased creatinine levels preoperatively ( odds ratio ( or ) 3.0 , p = 0.049 ) , history of atrial fibrillation ( af ) ( or 6.3 , p = 0.012 ) and high euroscore values ( or 2.6 , p = 0.017 ) had a significant greater probability to stay in the icu for more than 2 days . in addition , intraoperative hyperglycemia ( or 3.0 , p = 0.004 ) was strongly associated with longer icu - los . in conclusion , the high perioperative risk , the history of af and renal dysfunction , and the intraoperative hyperglycemia are significant predictors of prolonged icu stay . the early identification of patients at risk could allow the efficient icu resources ' allocation and the reduction of healthcare costs . this would contribute to nursing care planning depending on the availability of healthcare personnel and icu bed capacity . colorectal cancer is the third leading cancer worldwide . although incidence and mortality of colorectal cancer are gradually decreasing in the us , patients with metastatic colorectal cancer have poor prognosis with an estimated 5-year survival rate of less than 10% . over the past decade , advances in combination chemotherapy regimens for colorectal cancer have led to significant improvement in progression - free and overall survival . however , patients with metastatic disease gain little clinical benefit from conventional therapy , which is associated with grade 3~4 toxicity with negative effects on quality of life . in previous clinical studies , cell - based immunotherapy using dendritic cell vaccines and sentinel lymph node t cell therapy showed promising therapeutic results for metastatic colorectal cancer . in our preclinical and previous clinical studies , cytokine - induced killer ( cik ) cells treatment for colorectal cancer showed favorable responses without toxicities . here , we review current treatment options for colorectal cancer and summarize available clinical studies utilizing cell - based immunotherapy . based on these studies , we recommend the use cik cell therapy as a promising therapeutic strategy for patients with metastatic colorectal cancer .

the majority of swallowed indigestible foreign bodies pass through the gastrointestinal tract without complications [ 1 , 2 , 3 , 4 ] . however , there are three physiological narrowings involving the pylorus , duodenal c - loop and ileocecal valve . foreign bodies longer than 10 cm , such as a toothbrush , can not negotiate the duodenal c - loop due to its fixed retroperitoneal position . these objects should be endoscopically removed as soon as possible to avoid pressure necrosis and gastrointestinal perforation [ 5 , 6 , 7 ] . if endoscopic removal fails or there is evidence of obstruction or perforation , laparoscopic gastrotomy should be performed . an 18-year - old caucasian woman with no previous history of related medical problems was admitted to the department of internal medicine , division of gastroenterology , clinical hospital split because she had accidentally swallowed a toothbrush . the patient admitted she had been using the toothbrush to induce emesis . on presentation , 2 h after ingestion , she was asymptomatic and her vital signs were within normal limits . a plain abdominal x - ray study confirmed the presence of the foreign body in the left upper abdominal quadrant ( fig . informed written consent for upper gastrointestinal endoscopy was obtained from the patient and her parents . esophagogastroduodenoscopy revealed the toothbrush in the stomach with its head positioned against the gastric fundus . the extracted toothbrush was 20 cm long . repeated upper gastrointestinal endoscopy was performed 4 h later and showed no evidence of mucosal lesion to the stomach or the esophagus . the patient was discharged home in excellent clinical condition after being observed for 6 hours . foreign bodies in the stomach will pass uneventfully through the gastrointestinal tract in 8090% of cases [ 1 , 2 , 3 ] . however , foreign objects longer than 10 cm , such as a toothbrush , can not negotiate the duodenal c - loop due to its fixed retroperitoneal position . in such cases , these objects should be removed as soon as possible to avoid pressure necrosis and gastric perforation [ 2 , 3 , 4 , 5 ] . removal of long foreign bodies from the stomach is influenced by the patient 's clinical condition and technical abilities of the endoscopist [ 2 , 3 , 4 , 5 ] . if endoscopic removal fails or there is evidence of obstruction or perforation , surgical gastrotomy should be performed . we had no need for conscious sedation since the patient was actively participating during the procedure . special attention has to be paid during the extraction of the toothbrush to its alongside alignment with the esophagus . otherwise , this most critical and demanding part of the extraction procedure may easily result in mucosal damage or foreign body impaction . the second important phase of the extraction procedure is when the foreign body reaches the oropharynx . the patient has to extend his head backwards and the endoscopist has to reach for the toothbrush with his hand and pull it out . this case report describes a rare case in whom a toothbrush was safely extracted from the stomach endoscopically by snare extraction . the procedure is brief , does not require conscious sedation , and the patient can be discharged from hospital after a few hours . early endoscopic retrieval of the toothbrush is critical for reducing morbidity and mortality . in cases when endoscopic removal fails stress cardiomyopathy ( scm ) is an acute cardiac syndrome resembling st - segment elevation myocardial infarction characterized by decreased wall motion in the apical and midportion of the left ventricle ( lv ) . these abnormalities extend beyond a single coronary distribution without significant obstructive coronary artery disease.1 case reports in the literature suggest that strong psychological or physical stress is associated with the occurrence of scm.2 we believe that the present case is the first report of scm following diffuse alveolar hemorrhage ( dah ) owing to aspiration of polyethylene glycol ( peg ) . a 37-year - old woman was transferred from a primary clinic to our emergency department because of chest pain and shortness of breath . she had undergone gastroscopy and colonoscopy in the left lateral decubitus position at the clinic that day to evaluate her complaint of constipation . during the examination , she aspirated some gastric contents with residual peg . although she received immediate first aid , her hypoxia did not resolve fully and her chest pain gradually worsened . at the time of her arrival at the emergency department , she complained of chest pain , shortness of breath , and blood - tinged sputum . her initial vital signs were as follows : blood pressure , 80/50 mm hg ; heart rate , 114 beats / minute ; respiration rate , 22 breaths / minute ; body temperature , 37.2 ; and saturation , 95% in fio2 0.32 by pulse oxymetry . laboratory tests revealed a white blood cell count of 13,280/l with 94.3% neutrophils and 0.3% eosinophils , hemoglobin of 13.0 g / dl , platelet count of 182,000/l , activated partial thromboplastin time of 30.9 s , prothrombin time of 14.2 s , c - reactive protein of 2.10 mg / dl , significantly elevated troponin - i of 3.07 ng / ml , and elevated creatine kinase - mb of 15.7 ng / ml . the total creatine kinase level was not elevated at 194 u / l , but the creatine kinase - mb / creatine kinase ratio was markedly elevated at 8% . the electrocardiogram revealed sinus tachycardia without significant st segment abnormality or t - wave inversion . chest computed tomography showed ground - glass opacities and multiple low attenuation areas in the bilateral dependent portions , especially in the left upper lobe ( fig . the transthoracic echocardiogram revealed global hypokinesia , especially in the basal and midportions of the lv ( fig . the lv ejection fraction was estimated to be approximately 30% by the a / l method , and there was no lv outflow tract obstruction , pulmonary hypertension , or mitral regurgitation . initial pharmacologic management included aspirin , clopidogrel , and antibiotics based on the suspicion of acute coronary syndrome with aspiration pneumonia . although left ventriculography and ergonovine provocation tests were not conducted , the typical echocardiography findings without significant coronary artery lesions were sufficient to confirm the diagnosis of scm . after the patient was transferred to the cardiac intensive care unit , her symptoms of hypoxia and dyspnea gradually worsened and fresh hemoptysis accompanied this worsening . while she was on 80% oxygen , her arterial blood gas measurements were as follows : ph , 7.306 ; pco2 , 26.7 mm hg ; po2 , 58.4 mm hg ; hco3 , 13.0 mmol / l ; and sao2 , 88.2% . ultimately , the patient was intubated , ventilator care was begun , and fiberoptic bronchoscopy was performed . no bronchial mucosal lesion was identified , but five sequential aliquots of bonchoalveolar lavage ( bal ) fluid with a progressively bloody appearance were observed in the airway of the left upper area . analysis of the bal fluid revealed a red blood cell count of 28,000/mm and a white blood cell count of 100/mm with 40% neutrophils and 44% macrophages , consistent with alveolar hemorrhage . the patient received ventilator care for 3 days and was treated with methylprednisolone ( starting dose of 1 mg / kg and tapering for 7 days to stop ) and antibiotics . seven days into her hospitalization , her symptoms of dyspnea , hemoptysis , and chest discomfort had fully resolved . follow - up measurements of creatine kinase and creatine kinase - mb were normalized , and troponin - i was decreased to 0.13 ng / ml . the haziness on her chest radiograph had also disappeared ( fig . six months after her discharge from the hospital , a follow - up transthoracic echocardiogram revealed a 60% improvement in lv ejection fraction by the a / l method without regional wall motion abnormality ( fig . the pathophysiologic mechanism of scm has not been clearly established , but a catecholamine - mediated mechanism has been recognized as the most reliable precipitating factor.3 dah - induced hypoxia and hypercapnia have a multiplicative effect on the output of carotid chemoreceptors , and sympathetic activation is substantially increased.4 the exaggerated sympathetic responsiveness in dah triggers myocardial stunning , multi - vessel epicardial spasm , microvascular spasm , and direct catecholamine - mediated myocyte injury , each of which has been reported to cause scm.1 dah - induced scm could be explained in the same manner , but it has not previously been reported in the literature . dah refers to pulmonary hemorrhage originating from the alveolar capillaries , arterioles , and venules and is defined by the clinical symptoms of hemoptysis and anemia , diffuse radiographic pulmonary infiltration , and hypoxemic respiratory failure.5 dah is characterized histologically by the presence of intraalveolar red blood cells , fibrin , and hemosiderin - laden macrophages , which may take up 48 to 72 hours to accumulate.5,6 although a surgical biopsy specimen is considered the gold standard for diagnosis , it is often impractical . bal is an accepted alternative for confirming the diagnosis when 20% hemosiderin - laden macrophages are present.7 according to one recent study , however , the percentage of hemosiderin - laden macrophages in bal fluid can be increased in patients with dah , but this may not be diagnostic.6 in our case , we performed bal , but prussian blue stained hemosiderin - laden macrophages were not present in the bal fluid . bronchoscopic examination was performed within 24 hours , which may have been too early in the course of the disease to detect the accumulation of hemosiderin - laden macrophages . the area of alveolar hemorrhage in this case was not extensive and the clinical course of our patient was relatively mild compared with other reported cases of dah . injury to the alveolar microcirculation is mainly associated with local lung injury or systemic disorders such as vasculitis or connective tissue disease.5 our patient did not demonstrate any clinical evidence of a systemic disorder . therefore , it seems more likely that local lung injury due to aspirated fluid followed by progression of acute respiratory distress syndrome was the cause of the dah . although a wide range of dah causes have been reported on a case - by - case basis , peg exposure is a very rare etiology . peg is a popular bowel cleansing agent because of the safety and efficacy of the solution ; however , complications such as nausea , vomiting , and abdominal bloating are frequent.8 some studies have reported esophageal rupture with vomiting or respiratory failure after peg aspiration.9 - 11 the pathophysiological mechanism of peg - induced lung injury remains unclear . according to some studies , the hyperosmotic properties of peg solution can induce intra - luminary fluid shifting within the alveolar and interstitial spaces , resulting in pulmonary edema.12,13 in this case , massive lavage could retrieve a sufficient amount of aspirated fluid , especially peg , preventing further deterioration of chemical pneumonitis and acute respiratory distress syndrome . however , peg aspiration - induced dah in young adults is very rare and only a few patients have recovered fully after retrieval of aspirated fluid with bal .

most ingested foreign bodies will pass uneventfully through the gastrointestinal tract . nevertheless , long and rigid foreign bodies are associated with an increased risk of gastrointestinal impaction , perforation and bleeding . moreover , there has been no case of spontaneous passage of a toothbrush reported . therefore , the prompt removal of such ingested foreign objects is recommended before complications develop . this case report describes a case of an 18-year - old woman who accidentally swallowed her toothbrush . the toothbrush was successfully removed via flexible endoscopy using a polypectomy snare . a swallowed toothbrush is a special clinical challenge . early endoscopic retrieval of the toothbrush is critical for reducing morbidity and mortality . in cases when endoscopic removal fails , a laparoscopic surgical approach may be an alternative . stress cardiomyopathy ( scm ) is usually precipitated by a physiologically or psychologically stressful event . although it occurs only rarely , hypoxia- and hypercapnia - induced sympathetic activation may also cause scm . we present the case of a 37-year - old woman affected with scm after a routine colonoscopy . during the procedure , she aspirated residual polyethylene glycol from her stomach . hypotension , resting dyspnea , and hemoptysis were subsequently observed . laboratory findings revealed elevated cardiac enzymes , and a transthoracic echocardiogram revealed left ventricular ( lv ) global hypokinesia . she was ultimately diagnosed with diffuse alveolar hemorrhage - associated scm . after successful treatment with a ventilator and corticosteroids , her lv systolic function and dimensions normalized and she was discharged without complications .

solitary fibrous tumor ( sft ) is a rare neoplasm that was initially described in the visceral pleura and subsequently reported in extrapleural sites including the pericardium , peritoneum , mediastinum , lung , upper respiratory tract , thyroid , liver , testicle , nasal cavity , parotid , orbit , and meninges . sft development in the spine is an exceedingly rare event about which little is known . we present a unique case of an intradural extramedullary malignant sft having an extradural component with pulmonary metastases . to the best of our knowledge , this is the second case of malignant sft spine with distant metastases reported in literature . a 35-year - old female , with no comorbidities , presented with a 1 year history of progressive backache that was followed by lower extremity weakness for 4 months . neurological examination revealed paraparesis , hypertonia , increased deep tendon reflexes in the lower limbs , positive babinski sign , sustained bilateral ankle clonus and a sensory level of d12 with impaired temperature and proprioceptive sensation . contrast - enhanced magnetic resonance imaging ( mri ) of thoracolumbar spine revealed an intradural extramedullary mass ( 1.2 cm 1.25 cm ; which was predominantly hyperintense on t2 and hypointense on t1 images ) compressing and displacing cord at d10d11 level toward right side with extradural component ( 9.2 cm 5.2 cm ) extending into left neural foramen with widening at d10d11 level . contrast - enhanced computed tomography showed large well defined heterogeneously enhancing mass ( 9 cm 8.2 cm 6 cm ) in left paravertebral region extending from d10 to d12 vertebrae . the mass was seen passing through d10d12 neural foramina and causing its widening and extending into spinal canal causing rightward displacement of spinal cord [ figure 1c and d ] . she underwent d10d11 laminectomy with excision of intraspinal component of tumor by neurosurgeon from outside . histopathology examination ( hpe ) the tumor cells were positive for cd34 ( diffuse ) , cd99 , ema , bcl2 and negative for cd31 , s100 , cd117 , dog1 on immunohistochemistry ( ihc ) [ figure 2c and d ] . she was then referred to general surgery department of our institute where she was investigated for distant metastases which was negative , and then she underwent thoracotomy and excision of left paravertebral tumor . because of the incomplete surgical resection of the tumor and the high risk of locoregional and distant recurrence , adjuvant radiotherapy and chemotherapy was considered , but she went lost to follow - up for 5 months . when she turned up , an mri whole spine and positron emission tomography ( pet ) scan was performed for assessment of extent of disease . mri revealed a residual mass at the level of d11d12 neural foramina on left side ( 1.5 cm 1.2 cm 1.5 cm ) [ figure 3a and b ] with mild fluorodeoxyglucose uptake and multiple subcentimetric nodular lesions in bilateral lungs suggestive of metastases on pet scan . she was administered palliative radiotherapy of 20 gy in five fractions over 1 week to the primary residual site and planned for palliative chemotherapy with ifosfamide ( 1.4 g / m ; day 14 ) and epirubicin ( 60 mg / m ; day 12 ) . ( a ) sagittal view and ( b ) axial view of t2 weighted magnetic resonance imaging spine showing intradural extramedullary mass with extradural component . ( c ) axial view and ( d ) coronal view of contrast enhanced computed tomography scan showing left paravertebral mass ( a ) tumor is cellular and in diffuse growth pattern ( h and e , 100 ) . ( b ) tumor cells are round to oval with finely granular chromatin , indistinct cytoplasmic boundaries , and showing few mitosis ( h and e , 400 ) . ( d ) ki-67 proliferation index is ~5 - 7% ( a ) axial view and ( b ) sagittal view of t2 weighted magnetic resonance imaging of spine showing residual tumor sfts most often affect the pleura , but examples are increasingly being reported at other sites . spinal sfts are usually intramedullary ( 58% ) or intradural and extramedullary ( 24% ) . the majority of symptoms with which patients were presenting at time of admission were pain , hypoesthesia , paresis , urinary dysfunction , or a combination of these . similarly , as in our case , the symptoms consisted of a backache , paraparesis , and loss of sensation in lower extremities . 56% of sft patients are male , and most commonly it is seen on patients between 40 and 60 years old . two - thirds of sfts on mri were isointense on t1-weighted imaging with the remainder being either heterogeneous or hypointense . nearly , two - thirds of cases were hypointense on t2-weighted imaging with hyperintense being the next most common ( 17% ) . over three - quarters of cases demonstrated diffuse or homogeneous contrast enhancement with gadolinium administration ( 78% ) ; however , a significant portion demonstrated only partial or heterogeneous enhancement ( 21% ) . . typically , hpe features of sfts are spindle cells embedded in a fibrous matrix in a patternless architecture and alternating hypercellular and hypocellular areas with perivascular hyalinization or myxoid degeneration . overlapping histologic features , differentiation of sft from other soft tissue tumors may be difficult . typically , sfts are positive for cd34 , bcl-2 , and cd99 and negative for sma , desmin , pan - cytokeratin , and s-100 protein on ihc . findings such as nuclear atypia , increased cellularity , necrosis , and > 4 mitoses/10 hpfs , are suggestive of the malignant potential of sfts . in the current case , based on previous case reports , malignant sft showed rapid local recurrence and distant metastasis . at present , due to the rarity of the disease , standard therapies for malignant sft have not been well established . recurrence occurred commonly in cases involving incomplete excision , possibly caused by the level of difficulty in achieving complete resection . in these cases , . showed that conventional chemotherapy with anthracycline and ifosfamide is effective in controlling or stabilizing locally advanced and metastatic sfts . our patient being in metastatic setting , received palliative radiotherapy at local site and was considered for palliative chemotherapy with ifosfamide and epirubicin . the authors report here the second case of malignant sft of spine with distant metastases . although malignant sft is extremely rare , it should be considered in the differential diagnosis of spindle cell tumors in the spine . rufinamide ( 1-[(2,6-difluorophenyl)methyl]-1hydro-1,2,3-triazole-4 carboxamide ) is a novel antiepileptic drug ( aed ) that is structurally distinct from other aeds available on the market ( levy et al 2002a ) . an application was filed by eisai ltd in 2005 for european approval of this agent as an adjunctive treatment for seizures associated with lennox - gastaut syndrome ( lgs ) in children and adults ( eisai co ltd 2005 ) . lennox - gastaut syndrome is a catastrophic epileptic encephalopathy of childhood onset ( typically at age 35 years ) , for which effective treatment options are limited . this syndrome is associated with multiple types of generalized seizures , especially drop attacks and tonic seizures , delayed psychomotor development , behavioral and personality disorders , and a characteristic electroencephalogram pattern containing both generalized slow spike wave activity and paroxysms of generalized fast rhythmic activity during sleep which reflect excessive neocortical excitability ( markand 2003 ; nabbout and dulac 2003 ; guerrini 2006 ) . the long - term prognosis is very poor , with persistence of seizures in more than 75% of patients and severe mental retardation in more than 50% of patients ( markand 2003 ; dulac and engel 2003 ; schmidt and bourgeois 2000 ) . consequently , lgs is viewed as one of the most difficult epilepsies to treat . currently available treatment options for the epileptic encephalopathies in europe center around the use of valproate , with adjunctive benzodiazepines , topiramate , lamotrigine , or felbamate ( guerrini 2006 ) . however , a cochrane review has concluded that no single aed could be considered highly efficacious for lgs ( hancock and cross 2003 ) . indeed , the efficacy of valproate as a first - line treatment has been described as unimpressive , and its use in young children should be accompanied by great caution due to the risk of life - threatening hepatic toxicity ( schmidt and bourgeois 2000 ) . other first - generation aeds ( carbamazepine , phenytoin , and phenobarbital ) are not recommended for the treatment of lgs , due to the potential for aggravation of absence and myoclonic seizures by carbamazepine and phenytoin , and exacerbation of the behavioral problems seen in patients with lgs by barbiturates . among the benzodiazepines , clobazam may be viewed as the best tolerated , although there is no head - to - head comparison to confirm this . however , for this class of drugs , development of tolerance is well documented in the literature ( levy et al 2002b ) . the only aeds which have been evaluated in a randomized , controlled trial in lgs are lamotrigine , topiramate , and felbamate , the outcomes of which are discussed later in this paper . although felbamate is licenced for the treatment of lgs in the usa and some other countries ( eg , germany ) , its use has been limited following reports of serious toxicity ( borowicz et al 2004 ) . this has led to the recommendation that , in lgs , it should be used only in patients aged over 4 years who can not be treated satisfactorily with other aeds ( schmidt and bourgeois 2000 ) . thus , there is a clear need for new options in the management of this intractable condition . rufinamide was granted orphan drug status by the european medicines agency ( emea ) and the us food and drug administration ( fda ) in 2005 . this review will evaluate the relevant pharmacologic and clinical data for rufinamide , and explore its potential role in the future management of this severe form of epilepsy in the context of other products currently available for lgs . an extensive preclinical evaluation has suggested that the principal mechanism involved in the antiepileptic activity of rufinamide is its ability to modulate the activity of sodium channels , limiting high - frequency firing of neuronal action potentials over a broad range of concentrations , as demonstrated in vitro ( mclean et al 2005 ) . in radioligand binding studies , rufinamide does not interact with other neurotransmitter systems , including monoamine , adenosine , acetylcholine , histamine , glycine , -amino-3-hydroxy-5-methyl-4-isoxazole propionate ( ampa)/kainite , n - methyl - d - aspartate ( nmda ) , and -amino butyric acid ( gaba ) systems ( bialer et al 1996 ) . rufinamide has demonstrated broad spectrum anticonvulsant activity in animal models of epilepsy , elevating both electrically and chemically induced seizure thresholds and preventing seizure spread following oral and intraperitoneal administration in mice and rats ( white et al 2005 ) . furthermore , the protective index for rufinamide ( ratio of the concentration required for 50% anticonvulsant efficacy to that for neurotoxicity ) was superior to that of each of the comparator aeds examined ( phenytoin , phenobarbital , ethosuximide , and valproate ) . the pharmacokinetic properties of rufinamide have been established both in healthy volunteers and in patients with epilepsy . in a study of 3 healthy volunteers , an oral dose of 600 mg rufinamide demonstrated high absorption , and monoexponential elimination with a mean half - life ( t ) of 9 hours ( waldmeier et al 1996 ) . excretion was largely renal ( 85% ) and complete ( 98% ) within 7 days . following administration of 400 mg rufinamide as two 200-mg tablets or 400-mg oral suspension after a standard meal , mean maximum plasma concentrations ( cmax ) of 3.03 and 3.32 g / ml were reached after 6.6 and 7.2 hours , respectively , leading to elimination with a terminal t of 8.8 and 9.1 hours , respectively ( cheung et al 1995 ) . in a further study , the absorption of a single dose of 400 mg rufinamide was accelerated in fed subjects compared with fasting conditions , resulting in a 43% increase in exposure ( 96-hour exposure ) , without affecting the t ( cardot et al 1998 ) . however , no effect of food was observed with repeat dosing ( eisai , data on file ) , and no difference in rufinamide pharmacokinetics has been found between older ( aged 6677 years ) and younger ( 1840 years ) healthy subjects in either single- or multiple - dosing conditions ( chang et al 1998 ) . rufinamide pharmacokinetics in a study of 16 children aged between 2 and 17 years treated with 10 or 30 mg / kg daily rufinamide for 2 weeks were also comparable to those reported elsewhere for adult patients ( sachdeo et al 1998 ) . overall , evaluation of these data suggests that no age - related dose adjustments are likely to be required for either pediatric or elderly populations ( bialer et al 2001 ) . the pharmacokinetic parameters of single ascending doses of rufinamide ( 0 , 400 , 800 , 1200 mg ) have also been compared between 3 healthy subjects and 16 patients with epilepsy receiving enzyme - inducing aeds ( brunner et al 1994 ) . absorption rates were comparable , and both groups showed dose - related increases in cmax and exposure . rufinamide elimination was accelerated in aed - treated patients compared with healthy subjects , but t was independent of the dose administered in both groups . the pharmacokinetics of rufinamide have also been evaluated in 129 pediatric and adult patients with lgs ( critchley et al 2005 ) . rufinamide clearance was not affected by concomitant lamotrigine or topiramate , and was also unaffected by liver or kidney function . however , rufinamide concentrations were increased by concomitant valproate : 40% in children and 11% in adults . therefore , the rufinamide dosage may require adjustment during the addition or withdrawal of concomitant valproate therapy . rufinimade is extensively metabolized ( 2% excreted unchanged in urine and 2% in feces ) , predominantly via hydrolysis of the carboxylamide group to yield an inactive metabolite ( cgp-27292 ) , and is largely excreted in urine ( 84.7% of dose ) ( bialer et al 1999 ) . thus , rufinamide has a low propensity for drug drug interactions , based on its low protein binding ( approximately 34% ) and absence of metabolism via hepatic cytochrome p450 or inhibition of the major enzyme subclasses ( bialer et al 1996 ; kapeghian et al 1996 ) . this is supported by the absence of clinically relevant alterations of concomitant aed levels observed in a population pharmacokinetic drug this analysis was conducted using data from 5 double - blind studies involving pediatric and adult patients with inadequately controlled seizures despite previous treatment with up to 3 aeds . in total , 903 patients were receiving carbamazepine , 588 valproate , 200 lamotrigine , 299 phenytoin , 149 phenobarbital , and 69 topiramate . rufinamide co - administration did not affect the clearance of topiramate or valproate , but increased the clearance of carbamazepine and lamotrigine and decreased clearance of phenobarbital and phenytoin . however , all produced effects were small , were considered unlikely to be of clinical significance , and were independent of age . at a rufinamide plasma concentration of 15 g / ml , representing steady - state concentrations following doses of 45 mg / kg daily in children or 3200 mg / day in adults , changes in clearance conversely , in a similar study involving 471 patients , valproate co - administration decreased rufinamide clearance by approximately 22% , while any combination of phenytoin , phenobarbital , or primidone increased rufinamide clearance by approximately 25% , compared with patients not receiving these aeds ( bialer et al 2001 ) . rufinamide also resulted in a small increase in clearance of the oral contraceptive ortho - novum ( ortho - mcneil pharmaceutical , inc . raritan , nj , usa ) 1/35 in healthy female volunteers , though the clinical relevance of this effect is not known ( svendsen et al 1998 ) . the efficacy of rufinamide as an adjunctive therapy for treatment - resistant lgs has been evaluated in a multicenter , randomized , 12-week , double - blind , placebo - controlled , parallel - group study in 138 patients aged 437 years ( mean age 14.1 years ) , followed by an open - label extension phase ( glauser et al 2005a , 2005b ) . patients diagnosed with lgs , experiencing 90 seizures in the month prior to study entry , and receiving stable treatment with 13 concomitant aeds , were evaluated . after a 28-day baseline , patients randomized to twice - daily oral treatment with rufinamide ( n=74 ) or placebo ( n=64 ) entered a 14-day titration period ( rufinamide target dose 45 mg / kg daily ) , followed by 70 days of double - blind maintenance treatment . the reduction from baseline in the frequency of tonic - atonic seizures ( drop attacks ) per 28 days was significantly greater with rufinamide than placebo ( median change 42.5% vs + 1.4% , respectively , p<0.0001 ) . similarly , the reduction from baseline in total seizure frequency per 28 days was significantly greater with rufinamide than placebo treatment ( median change 32.7% vs 11.7% , respectively , p=0.0015 ) . these improvements were associated with significantly greater proportions of responders ( patients achieving 50% reduction in seizures per 28 days ) with rufinamide than placebo treatment , both for tonic - atonic seizures ( 42.5% vs 16.7% , p=0.0020 ) ( glauser et al 2005a ) and total seizure frequency ( 31.1% vs 10.9% , p=0.0045 ) ( kluger et al 2006 ) . following completion of the double - blind study , 123 patients continued to receive open - label treatment in the extension phase , at a median dose of 1800 mg / day ( range 1034865 mg / day ) for a median duration of 432 days ( range 101149 days ) ( glauser et al 2005b ) . the reduction in median total seizure frequency observed at 12 weeks was maintained , with some further improvement noted with continued treatment for up to 3 years ( see figure 1 ) . similarly , the responder rate for total seizures was maintained , with 36.9% of patients achieving a reduction in total seizures of at least 50% overall during the extension study . furthermore , 21.3% of patients achieved a 75% reduction in total seizures overall during the extension study . during the double - blind study of rufinamide in patients with lgs , the most common adverse events ( reported by 10% patients in either treatment group ) were : somnolence ( 24.3% rufinamide , 12.5% placebo ) , vomiting ( 21.6% rufinamide , 6.3% placebo ) , pyrexia ( 13.5% rufinamide , 17.2% placebo ) , and diarrhea ( 5.4% rufinamide , 10.9% placebo ) ( glauser et al 2005a ) . notably , rufinamide treatment was associated with a lower incidence of cognitive / psychiatric adverse events ( such as psychomotor hyperactivity and lethargy ) than placebo treatment ( 17.6% vs 23.4% , respectively ) . there was no clinically significant change in clinical laboratory tests , physical examinations or vital signs . during the subsequent open - label extension study , the most commonly reported all - causality adverse events were vomiting ( 30.6% ) , pyrexia ( 25.8% ) , upper respiratory tract infection ( 21.8% ) , and somnolence ( 21.0% ) ( glauser et al 2005b ) . there were two deaths , which were not considered to be related to rufinamide treatment . thus , long - term treatment of patients with lgs does not appear to be associated with any increase in central nervous system ( cns ) adverse events , with somnolence noted with somewhat lower frequency than during the double - blind phase . these tolerability and safety data in patients with lgs are further supported by a pooled analysis of rufinamide tolerability and safety in a large population of epilepsy patients , in which the data were evaluated separately for patients receiving short - term or long - term treatment ( krauss et al 2005 ) . the short - term safety population comprised 1240 patients treated with rufinamide ( mean age 31.7 years ; mean rufinamide dose 1373 mg / day ; mean duration of treatment 2.8 months ) and 635 placebo recipients ( mean age 28.6 years , mean duration of treatment 3.0 months ) . somnolence was reported somewhat less frequently in this population than in the lgs study , with an incidence of 11.8% for rufinamide and 9.1% among placebo recipients . the timing of events in this pooled analysis indicated a pattern of early onset and rapid resolution , and a tolerability profile not substantially different from placebo ; the incidence of the most frequently reported adverse events with rufinamide vs placebo were headache ( 22.9% vs 18.9% ) , dizziness ( 15.5% vs 9.4% ) , fatigue ( 13.6% vs 9.0% ) , somnolence ( 11.8% vs 9.1% ) , and nausea ( 11.4% vs 7.6% ) . the pooled tolerability and safety analysis included 1978 patients assessed for long - term tolerability and safety ( mean age 31.3 years , mean rufinamide dose 1700 mg / day , maximum dose 7200 mg / day ) , 47% of whom were treated with rufinamide for at least 12 months ; the most frequently reported adverse events in this population were headache ( 29.5% ) , dizziness ( 22.5% ) , and fatigue ( 17.7% ) ( krauss et al 2005 ) . few randomized , controlled clinical trials have been conducted in patients with lgs , none of which is a head - to - head comparison . consequently , the available treatments for lgs can not be compared directly , and only limited conclusions may be drawn from indirect comparisons . the most robust approaches for indirect comparisons include systematic reviews and meta - analyses , such as those conducted by the cochrane group . a systematic ( cochrane ) review of randomized , controlled trials in lgs ( hancock and cross 2003 ) retrieved only 5 evaluable studies : one each conducted with topiramate , lamotrigine , felbamate , cinromide , and a thyrotropin releasing hormone ( trh ) derivative , respectively ( anonymous 1989 , 1993 ; inanaga et al 1989 ; motte et al 1997 ; sachdeo et al 1999 ) . this review concluded that no single agent was found to be highly efficacious in lgs , and that of those evaluated only lamotrigine , topiramate and felbamate were considered potentially efficacious as add - on therapy ( hancock and cross 2003 ) . data derived from this cochrane analysis for each of the clinical trials are summarized in table 1 , together with data from the more recently reported rufinamide study ( glauser et al 2005a ) . although this provides an indirect comparison , it is of interest to note that rufinamide produced a particularly marked effect in the reduction in drop attacks ( tonic - atonic seizures ) , since this is also the parameter viewed as the primary outcome variable for lgs according to guidelines from the american academy of neurology ( french et al 2004 ) . the median reduction in tonic - atonic seizures was 42.5% with rufinamide , which compares well with rates observed in the separate studies with topiramate ( 14.8% reduction ) or lamotrigine ( 34% reduction ) ; the effect of felbamate on drop attacks was not reported . when corrected for the magnitude of placebo response , the median effect size for rufinamide on drop attacks was 41.1% , compared with 19.9% for topiramate and 14.8% for lamotrigine . similarly , the proportion of patients who could be considered responders to therapy ( achieving at least a 50% decrease in drop attacks ) was 42.5% with rufinamide , 45.6% with topiramate , and 37.3% with lamotrigine , or 25.8% , 25.6% , and 14.8% , respectively , when corrected for placebo response . as with drop attacks , the effects of these agents on total seizure frequency produced some interesting findings . the rufinamide , topiramate , and lamotrigine studies demonstrated 32.7% , 20.6% , and 32.0% median reductions in total seizures , and treatment effect sizes ( adjusted for placebo ) of 20.0% , 12.6% , and 17.6% , respectively . in the felbamate study , outcomes were reported as mean rather than median values , resulting in a 19% mean reduction in total seizures , and a 23% mean effect size . the number of patients achieving complete cessation of seizures has only been reported with felbamate , and although these results are favorable , can not be compared with the other aeds in the studies available to date . of course , the overall benefit of treatment comprises a balance between reduction in ( or cessation of ) seizures and tolerability / safety . the reported tolerability of treatment was broadly comparable among the studies described in table 1 , with relatively small differences in rates of discontinuations due to adverse events . few randomized , controlled clinical trials have been conducted in patients with lgs , none of which is a head - to - head comparison . consequently , the available treatments for lgs can not be compared directly , and only limited conclusions may be drawn from indirect comparisons . the most robust approaches for indirect comparisons include systematic reviews and meta - analyses , such as those conducted by the cochrane group . a systematic ( cochrane ) review of randomized , controlled trials in lgs ( hancock and cross 2003 ) retrieved only 5 evaluable studies : one each conducted with topiramate , lamotrigine , felbamate , cinromide , and a thyrotropin releasing hormone ( trh ) derivative , respectively ( anonymous 1989 , 1993 ; inanaga et al 1989 ; motte et al 1997 ; sachdeo et al 1999 ) . this review concluded that no single agent was found to be highly efficacious in lgs , and that of those evaluated only lamotrigine , topiramate and felbamate were considered potentially efficacious as add - on therapy ( hancock and cross 2003 ) . data derived from this cochrane analysis for each of the clinical trials are summarized in table 1 , together with data from the more recently reported rufinamide study ( glauser et al 2005a ) . although this provides an indirect comparison , it is of interest to note that rufinamide produced a particularly marked effect in the reduction in drop attacks ( tonic - atonic seizures ) , since this is also the parameter viewed as the primary outcome variable for lgs according to guidelines from the american academy of neurology ( french et al 2004 ) . the median reduction in tonic - atonic seizures was 42.5% with rufinamide , which compares well with rates observed in the separate studies with topiramate ( 14.8% reduction ) or lamotrigine ( 34% reduction ) ; the effect of felbamate on drop attacks was not reported . when corrected for the magnitude of placebo response , the median effect size for rufinamide on drop attacks was 41.1% , compared with 19.9% for topiramate and 14.8% for lamotrigine . similarly , the proportion of patients who could be considered responders to therapy ( achieving at least a 50% decrease in drop attacks ) was 42.5% with rufinamide , 45.6% with topiramate , and 37.3% with lamotrigine , or 25.8% , 25.6% , and 14.8% , respectively , when corrected for placebo response . as with drop attacks , the effects of these agents on total seizure frequency produced some interesting findings . the rufinamide , topiramate , and lamotrigine studies demonstrated 32.7% , 20.6% , and 32.0% median reductions in total seizures , and treatment effect sizes ( adjusted for placebo ) of 20.0% , 12.6% , and 17.6% , respectively . in the felbamate study , outcomes were reported as mean rather than median values , resulting in a 19% mean reduction in total seizures , and a 23% mean effect size . the number of patients achieving complete cessation of seizures has only been reported with felbamate , and although these results are favorable , can not be compared with the other aeds in the studies available to date . of course , the overall benefit of treatment comprises a balance between reduction in ( or cessation of ) seizures and tolerability / safety . the reported tolerability of treatment was broadly comparable among the studies described in table 1 , with relatively small differences in rates of discontinuations due to adverse events . each of these currently available agents has been associated with warnings relating to severe adverse events . the use of felbamate has been restricted in many countries following reports of idiosyncratic aplastic anemia and hepatic failure , which led to its initial withdrawal in the usa , although limited use was granted subsequently ( fda 1994 ) . similarly , the efficacy of lamotrigine must be balanced against the risk of serious skin reactions in susceptible individuals , for which boxed warnings or safety alerts have been issued ( committee on safety of medicines 1996 ; fda 1997 ) , while the use of topiramate is associated with warnings relating to oligohydrosis and hyperthermia in children , and also metabolic acidosis and glaucoma ( fda 2003 ; janssen - cilag 2006 ) . in addition , factors such as cognitive slowing and weight loss may be issues with the use of topiramate ( levy et al 2002c ) . the timing of these warnings , some time after the initial marketing of these aeds , illustrates the difficulties in identifying such events during clinical trials , and that further evaluation is required to confirm the long - term safety of rufinamide in comparison with the marketed drugs . indeed , increased qt interval dispersion has been demonstrated in children with epilepsy , potentially predisposing them to serious cardiac events ( akalin et al 2003 ) . rufinamide has demonstrated excellent cardiovascular tolerability , with no increase in qt intervals in a cardiac safety study conducted in 15 healthy volunteers treated with placebo or ascending doses of rufinamide ( from 800 to 7200 mg / day ) over 18 days ( marchand et al 2005 ) . on the contrary , lgs is a catastrophic and debilitating age - specific epileptic disorder , for which a substantial unmet need exists for highly effective treatment options . the data reviewed here indicate that adjunctive therapy with rufinamide , a new aed that is structurally unrelated to other currently available aeds , significantly reduces seizure frequency in patients with lgs , accompanied by good tolerability , both of which are maintained during long - term use . in particular , the reduction in drop attacks adds substantially to patient quality of life . the efficacy of rufinamide is most likely derived from its ability to block sodium channels , thereby limiting high - frequency action potential firing , a mechanism shared by some other aeds including phenytoin , carbamazepine , lamotrigine , and oxcarbazepine ( mclean et al 2005 ) . however , it is commonplace for patients with lgs to remain symptomatic despite optimal therapy , and it is notable that , in the studies reviewed , adjunctive rufinamide treatment was able to exert significant improvement in a treatment - resistant population receiving 13 other aeds at baseline . in an indirect comparison with randomized controlled trials examining other aeds , the results with rufinamide appear impressive : a median reduction in drop attacks of 42.5% with rufinamide , compared with 14.8% with topiramate and 34% for lamotrigine , representing treatment effect sizes , after adjustment for placebo response , of 41.1% , 19.9% , and 14.8% , respectively . this was associated with a median reduction in total seizures of 32.7% with rufinamide , compared with 20.6% in the topiramate study and 32% in the lamotrigine study . although the full safety data for the rufinamide study in lgs have yet to be reported , data from a pooled safety database suggest a good tolerability profile for both short- and long - term use of rufinamide , with a pattern of early onset and early resolution of events . cardiac safety profile , based on a dose - ranging study in healthy volunteers which included qt analysis . however , the full spectrum of efficacy as well as the safety profile of rufinamide will become clear with accumulation of long - term clinical experience and usage . overall , these findings suggest that rufinamide may be a valuable option for the adjunctive treatment of lgs .

solitary fibrous tumor ( sft ) usually originates from the pleura because of abnormal proliferation of fibroblast cells . it is extremely rare for the tumor to originate from the spine . here , we report the second case of malignant sft of thoracic spine with distant metastases in a 35-years - old female . rufinamide , a triazole derivative that is structurally distinct from currently marketed antiepileptic drugs ( aeds ) , is in development for the adjunctive treatment of lennox - gastaut syndrome ( lgs ) in children and adults . rufinamide is well absorbed after oral administration , demonstrates low protein binding , and is metabolized by enzymatic hydrolysis without involvement of cytochrome p450 enzymes , conferring a low drug interaction potential . in a randomized , double - blind trial involving 138 adult and pediatric patients with lgs , compared with placebo , rufinamide 45 mg / kg / day resulted in significantly superior reductions in drop attacks ( median change 42.5% vs + 1.4% with placebo ) and total seizures ( 32.1% vs 11.7% with placebo ) , accompanied by significantly higher responder rates . these results are comparable with findings reported for other aeds in randomized , controlled clinical trials in patients with lgs . rufinamide produced statistically significant seizure reduction which was maintained during long - term therapy and accompanied by good tolerability . the most frequently reported adverse events from a pooled safety database evaluating short- and long - term therapy were headache ( 22.9% and 29.5% ) , dizziness ( 15.5% and 22.5% ) and fatigue ( 13.6% and 17.7% ) . rufinamide therefore presents a favorable efficacy and tolerability profile and is a promising candidate for the adjunctive therapy of lgs .

a 55-year - old man presented at our emergency department with sudden - onset sharp chest pain that had started two hours previously . he also complained of progressive swelling and bruising on his neck , and the development of dysphagia and dyspnea over the previous two hours . he had a history of admission with spontaneous hemothorax seven years previously , and at that time , despite several work - ups for hemothorax , its origin was not found . other than this incident , he had hypertension but no history of gastrointestinal or bleeding disorders . upon arrival at the emergency department extensive neck swelling was observed , as well as bruising on the neck and upper chest . during the examination , he suddenly vomited bright red blood with food material three times , and approximately a quarter - cup of blood was generated on each occasion . other laboratory tests , including cardiac markers and his coagulation profile , were within their normal ranges . 1b ) revealed extensive non - enhancing soft tissue lesions in the posterior mediastinum and neck , which were suspected to be hematomas . emergency gastroscopy showed many blood clots but no definite focus of gastrointestinal bleeding , such as an ulcer , mass , or perforation . instead , gastroscopy showed extrinsic esophageal compression . since the patient was hemodynamically stable , conservative management was adopted in the intensive care unit . followup ct on the third day post - admission showed a focal aneurysm in the right bronchointercostal trunk ( fig . subsequent selective bronchial arteriography revealed a 1-cm aneurysm in the right bronchial artery ( fig . since the aneurysm was considered to be the cause of the hematemesis and mediastinal hemorrhage , we decided to perform embolization to prevent recurrence . the right bronchial artery was subsequently embolized with four microcoils ( vortx-18 ; boston scientific co. , marlborough , ma , usa ) and 33% glue ( fig . 2c ) , and repeat ct showed that the mediastinal hematoma had nearly disappeared ( fig . the patient was discharged without complications . over the course of one year of follow - up spontaneous mediastinal hemorrhage can develop as a result of trauma , aortic dissection , the valsalva maneuver , or iatrogenic procedures . the rupture of a bronchial artery aneurysm ( baa ) is also known to result in mediastinal hemorrhage . when a baa ruptures into the mediastinum , most patients present with chest pain , hemothorax , or hemomediastinum . furthermore , if hematemesis is a prominent symptom of baa rupture , it can be confused with boerhaave s syndrome , variceal disease , or a perforated ulcer . to the best of our knowledge , only three cases of baa presenting with hematemesis have been reported in the literature [ 46 ] . in one case , a pinhole connection between the aneurysm and esophagus was found during an endoscopic examination , but in the present case , no evidence of communication was found between the aneurysm and the esophagus . we first suspected that the mediastinal hemorrhage and hematemesis were caused by the perforation of an esophageal ulcer , but repeated gastroscopy showed a normal esophagus . the only abnormal finding was the finding of a baa without evidence of extravasation three days after the onset of symptoms . after ruling out the possibility of mediastinal hematoma , the baa was the only remaining possible source of the mediastinal hemorrhage . since the baa was regarded as the source of the mediastinal hemorrhage and hematemesis , it was embolized to avoid recurrence . the treatment of a ruptured baa depends on the patient s hemodynamic status and the presence of cardiorespiratory compromise . if the patient is hemodynamically stable , endovascular embolization is considered to be the first - line management strategy , and surgery should only be considered when embolization is contraindicated , as in patients allergic to contrast medium or when a medullary artery is involved . in patients presenting at an emergency department with hematemesis and mediastinal hemorrhage , these include isolates from disease outbreaks in africa , europe , the caribbean , and , more recently , from the russian / trans - caucasian epizootic and attenuated variants . in brief , swine red bone marrow cell culture was used for asfv isolate amplification , and swine anti - asfv serum and erythrocytes were subsequently added to the culture . asfv isolates for which the hemadsorption phenomenon was inhibited by serum belonging to the same group within serogroups 18 were clustered into a homologous serogroup . only asfv hemadsorbing strains could be analyzed by this method , and some hemadsorbing asfv isolates could not be placed into existing serogroups because hai was not observed with available reference serum ( 8,9 ) . asfv isolates from the depository at vniivvim were also classified by using a standard asfv genotyping protocol previously published by bastos et al . , the variable part of the p72 ( b646l ) gene was amplified by conventional pcr , and the amplicons were directly sequenced by using a 3130xl genetic analyzer ( applied biosystems , foster city , ca , usa ) according to manufacturer s recommendations . sequences determined at vniivvim were aligned with other publically available asfv sequences and analyzed by using minimum evolution ; a rooted tree was constructed with mega 5.0 software ( 14 ) and edited with figtree v1.4 ( http://tree.bio.ed.ac.uk/ ) ( figure 1 ) . phylogenetic tree of african swine fever virus ( asfv ) isolates maintained in a collection at the national research institute for veterinary virology and microbiology in okrov , russia ; the variable part of b646l gene relative to the 22 known p72 genotypes ( labeled i - xxii ) was used for analysis . the tree was reconstructed by using the minimum evolution method with 1,000 replicates . * the isolates were selected from the african swine fever virus ( asfv ) collection at the national research institute for veterinary virology and microbiology ( vniivvim ) in okrov , russia . asfv genotypes are assigned according to p72 ( b646l ) nucleotide sequencing and phylogenetic tree reconstruction . serogroups are defined on the basis of results of hemadsorption inhibition assay ( hai ) with reference serum ( serogroups 18 ) . democratic republic of congo ; , not applicable ; nk , not known ; nd , not defined . if a country name changed since the virus was isolated , the current name of the country is shown in parentheses . isolates bartlett , magadi , and davis could not be placed into an existing serogroup because no hai was observed with available reference serum , so exact serogroups for these isolates are not defined . isolate spenser does not belong to any of the 22 known genotypes and remains untyped . newly identified asfv genotypes and known serogroups were mapped together so their geographic distribution in africa and europe ( including the european part of the russian federation ) could be visualized . the results ( figure 2 ) show that genotypic and serogroup diversity are greatest in a relatively limited area , mainly in southeastern africa . in contrast , non - asfv endemic countries , where asf outbreaks were caused by asfv of a single genotype , exhibited low or no serogroup diversity . world distribution of african swine fever virus ( asfv ) isolates maintained in a collection at the national research institute for veterinary virology and microbiology in okrov , russia . results of p72 genotyping and hemadsorption inhibition assay of asfv isolates are summarized on the map . genotype ii of asfv isolates from lithuania , latvia , estonia , poland , and belarus was identified by cisa - inia ( animal health research center ; european union reference laboratory for african swine fever ) . asfv isolate o-77 , which was isolated in 1977 from odessa , ukraine ( at the time , part of the union of soviet socialist republics ) , was used in this study . on the basis of cisa - inia results , currently circulating isolates in ukraine belong to genotype ii . the oval with a 1 inside indicates switzerland ; the oval with a 2 inside indicates so tome and principe . country names are presented as 2-letter country codes as designated by the international organization for standardization country codes ( iso 3166 , http://www.iso.org/iso/country_codes.htm ) . single genotype clades of asfv were observed to contain viruses of multiple serogroups ( table ) . for example , asfv isolates belonging to serogroups 1 , 2 , and 4 were specifically clustered within genotype i , and did not group with other genotypes . we also found several serogroups of asf viruses within genotype x. the asfv isolates tsp80 ( serogroup 5 ) and ts-7 ( serogroup 6 ) were subsequently isolated from 1 field sample derived from a naturally infected pig in tanzania . of note , 1 serogroup 2 isolate ( spenser ) demonstrated a novel genotype within the p72 phylogenetic tree and relative to other serogroup 2 viruses ( figure 1 ) , indicating that the p72 genotype , in addition to lacking serotype resolution , has potential to be incongruous relative to serogroup . together , these data indicate that the antigenic heterogeneity of asfv strains is not fully captured by using the standard genotyping approach . the virus elements responsible for protective and cross - protective immune responses are not well known . given the structural and genetic complexity of asfv , it is likely that genes encoding different antigens will be more suited for virus typing . substantial genetic variability can exist between strains and predominate in specific genomic regions , and it is these regions that may provide improved targets for genotyping . our findings support that of a previous study that showed that geographic areas with asfv of high genotypic and serotypic diversity are located in countries where multiple mechanisms of asf transmission ( mixed sylvatic and domestic cycle ) are established ( 11 ) . hai serology provides a measure of asfv typing that , compared with p72 genotyping , better discriminates biologically pertinent phenotypes . viruses belonging to one p72 genotype may be serotypically heterogeneous : strains that are closely related genetically , even from a single isolate , may have different phenotypes and form homologous serogroups . our assessment of asfv genotyping relating to hai serotyping shows the serologic diversity within a p72 genotype . our results highlight the potential for using serogroup classification to understand issues of homologous cross - protection among asfv isolates and virus determinants that influence disease emergence . the key finding from our study is that p72 asfv phylogenetic analysis fails to accurately define asfv hai serogroups . thus , conventional asfv genotyping can not discriminate between viruses of different virulence or predict the efficacy of a specific asfv vaccine . this finding also suggests that for vaccination - based control of asf , it may be more important to determine serogroups rather than genotypes of asfv isolates .

hematemesis is a rare manifestation of a ruptured bronchial artery aneurysm ( baa ) in the mediastinum . it is difficult to diagnose a ruptured baa presenting as hematemesis , because it can be confused with other diseases , such as boerhaave s syndrome , variceal disease , or a perforated ulcer . in this report , we describe a case of baa resulting in hematemesis and mediastinal hemorrhage . african swine fever virus ( asfv ) causes highly lethal hemorrhagic disease among pigs , and asfv s extreme antigenic diversity hinders vaccine development . we show that p72 asfv phylogenetic analysis does not accurately define asfv hemadsorption inhibition assay serogroups . thus , conventional asfv genotyping can not discriminate between viruses of different virulence or predict efficacy of a specific asfv vaccine .

entrapment of the ulnar nerve in the elbow region is the second most common compression neuropathy in the upper extremity . many procedures have been advocated for decompression of the ulnar nerve at the elbow , including anterior subcutaneous transposition , anterior intramuscular transposition , anterior submuscular transposition , medial epicondylectomy , simple decompression , neurolysis , and in situ decompression . the introduction of endoscopic release , the newest of all the surgical options for this problem has been described by several authors . the new approach to peripheral nerve surgery specifically in cubital tunnel syndrome is the introduction of the endoscopic procedure . since the advent of endoscopic methods to release the course of the ulnar nerve entrapment at the elbow site , there has been a flurry of interest and controversy about the efficacy and safety of this newest technique . endoscopic decompression of the ulnar nerve at the elbow was first described in 1995 by tsai et al . . there are different variations of endoscopic surgical technique , but the purpose and goals preserve the vascularity of the ulnar nerve at the elbow , release all possible compression sites , allow early mobilisation of the elbow , avoid extensive surgical exposure , and scar discomfort . it provides for limited soft tissue dissection , thereby allowing more rapid recovery with minimal scarring . potential ulnar nerve entrapment can occur at five sites around the elbow : the arcade of struthers , the medial intermuscular septum , the medial epicondyle , the cubital tunnel , and the deep flexor pronator aponeurosis . various surgical techniques for decompression of the ulnar nerve have been described in the literature , and a definitive gold standard does not exist . comparative studies have shown some short - term advantages to one or another technique , but overall results between the treatments have essentially been equivocal . a thorough preoperative diagnosis and workup will help guide us for the type of surgical technique . we report our experience with this newer technique in six patients , with special focus on the clinical and surgical outcome . the study was carried out in the department of orthopaedics and traumatology , division of hand and foot surgery , hong kong university medical centre at queen mary hospital from period of february 2008 to may 2010 . their data was retrieved from queen mary hospital and david trench rehabilitation centre . there were six cases with endoscopic cubital tunnel release . the inclusion criteria are failure of conservative treatment for at least 6 months , have progressive clinical findings of atrophy , elevated two - point sensory discrimination , weakness of the ulnar nerve distribution , and positive electrophysiological conformation studies . the exclusion criteria include the subluxation of the ulnar nerve if it was felt to subluxate over the medial epicondyle on passive elbow flexion , long - standing elbow contracture , mass or space - occupying lesions , history of significant trauma of the elbow , history of cervical neuropathy or the double crush syndrome , prior surgery , scarred , adherent nerve , history of fracture of the elbow malunion , and cubital valgus . all patients underwent conservative management with splintage , avoidance of provocative activities , and rehabilitation program . failure of conservative management and positive electrophysiological studies with clinical weakness of the ulnar nerve distribution and/or muscle atrophy is the criteria in surgical management . all six patients were offered endoscopic release as the alternative for standard open release procedure . a uniform endoscopic cubital tunnel release was done with a single senior orthopaedic hand surgeon with experience in endoscope release technique . six patients who had compressive cubital tunnel syndrome at the elbow were treated with endoscopic cutr at our institution after electrophysiological conformation of the diagnosis . the sensory conduction studies ( i.e. , amplitude of the sensory action potential and sensory conduction velocity of the ulnar nerve ) were considered prolonged in all cases ; the motor conduction studies ( i.e. , nerve site , onset , amplitude , segment , latency difference , distance , and conduction velocity ) were prolonged in all cases . the dellon 's scale score was used for rating the severity of the lesions and the postoperative outcome was assessed based on the modified bishop rating scale system . the final postoperative outcome was assessed 6 months after the surgery by subjective information based on modified bishop scoring classification system ( severity of residual system , improvement , work status , strength , and sensibility ) , this bishop score is defined as poor , 0 to 2 ; fair , 3 to 4 ; good , 5 to 7 ; excellent , 8 to 9 . surgery is performed in patient under ga in supine position with upper limb side table . the patient shoulder in abduction and external rotated and elbow slightly extended in position . a pneumatic tourniquet applied in the most proximal area of the arm , a clearly marked of anatomical portal and course of the ulnar nerve . a 1.5 cm3 cm transverse incision is made between the medial humeral epicondyle and the olecranon at the course of the ulnar nerve . the ulnar nerve has been identified , subcutaneous pouch developed and endoscope set inserted ( karl storz , tuttlingen , germany ) into subcutaneous tunnel and release under endoscopic guidance proximally . another distal pouch developed with the same entry site , and endoscope has been inserted . a distal release into two heads of flexor carpi ulnaris and the nerve branches of the fcu have been identified and protected . saline irrigation and drain was inserted and anchored in view of oozing from minor vessels and clot formation . a soft dressing is applied with long - arm backslab in slightly 45 degrees elbow extension as haemostasis for two days , and gentle active mobilisation exercise . our patient population in this series is six patients : two woman and four men with 1 : 2 being the ratio . the median average age was 55 years ( range , 3377 ) . a surgical endoscopic release was performed on the right side in four elbows and on the left side in two elbows . the main job profile of two patients is mainly table - top tasks , one works as dental hygienist , and three are nonworking in their retirement age , and all patients were not manual labourer . in five patients , the right side is dominant except one patient who was ambidextrous ( table 1 ) . the mean length of the surgery in the endoscopic release average 47 minutes ( range , 3262 minutes ) . the mean length of the skin incision was 2.25 cm ( range , 1.53 cm ) . retrospectively , no patient was classified as mild , three patients ( 50% ) were moderate , and three ( 50% ) were severe according to dellon 's classification in stages of ulnar nerve compression at the elbow ( table 2 ) . the postoperative outcome result is based on modified bishop rating system classification based on severity of residual symptoms , improvement of symptoms , work status , strength , and sensitivity which shows two ( 33% ) with excellent results , and four ( 66% ) have good results ( table 3 ) . the dental hygienist who has major risk factors identified sustained exposed to work related repetitive left elbow flexion more than 90 degrees constantly during the therapy session ( > 1 hour ) and sustained holding of hand tools . surgical site is not the dominant hand , then exposure to the vibratory tooth scaler machine is not a risk factor . there is no change of job to this patient , but with recommendations incorporate half - hour sessions of duties avoiding sustained gripping or sustaining affected elbow flexion > 90 degrees after every half - hour of therapy session and regular intermittent stretching of left elbow during the therapy session . three patients who are in retirement age with initial subjective evaluation base on modified bishop 's classification reach to score of good result without adding the subtype point score of work status of the patients for the reason , and this is not applicable to this group of patients ( table 4 ) . none of the patient was converted endoscopic release to in situ open due to any complication . no patients develop a postoperative infection , cutaneous nerve injury , hematoma , or painful surgical site . all patients improved symptom one day after the surgery with sensory loss improved and go back to their full activity in one month , and three patients previously went back to work after two months . the recovery and return to work was rapid and with a high patient satisfaction and no recurrence of symptom noted . none of the patients complained about scar discomfort , painful neuroma , burning sensation , superficial hypersensitivity , no elbow extension deficit , or ulnar nerve subluxation . sensory lost improved in all patients after the surgery and gradually improve after reevaluation at six - month subjective scale with good to excellent results . all preoperative electrophysiological studies were considered in all cases with abnormal results and with postoperative comparison which result findings five ( 83% ) residual changes but one patient ( 16% ) who has residual impaired because of the preoperative findings of evidence of axonal loss . this patient presented with severe preoperative compromise of the intrinsic musculature of the hand and subjective persistent numbness of the ulnar 1(1/2 ) side digits distribution , the postoperative ncv shows evidence of axonal loss but after 6 months postoperative ncs shows interval improvement with moderate prolonged . this patient improved the subjective parameters of modified bishop scoring system to good score result even when with persistent ulnar nerve distribution numbness and subjectively claimed that numbness decrease by 90% postoperatively , and objective parameter was satisfactory with overall clinical improvement . overall , these six cases were good to excellent subjective improvement of the result and also objective parameters in grip strength improvement . patients usually complain of sensory symptoms rather than muscle weakness so the result of the surgery was considered satisfactory by the patients , as their major complaints were related to the sensory symptoms . there were no complications in this series of six patients with good patient satisfaction and successful outcome without untoward complication . there is no gold standard in the surgical management of the cubital tunnel syndrome for the main reason that no single standard consensus of primary problem in nerve compression . in two groups of authors one believed that nerve compression is caused by overlying structure and that the syndrome is best treated by decompression of the ulnar nerve without removing it from its bed . other group of authors are citing evidence that the nerve is under tension with elbow flexion . that can only be relieved by placing the nerve anterior to the medial epicondyle . various surgical techniques for decompression of the ulnar nerve have been described in the literature , and a definitive gold standard does not exist . heithoff ( 1999 ) stated that all surgical techniques for cubital tunnel syndrome yielded similar results and that the choice of surgical technique should be based on simplicity . the endoscopic approach to in situ decompression in our series of six patients has a rapidity of postoperative improvement of symptoms compatible to the study of hoffmann and siemionow in 2006 . a completely new approach to surgery which enables to see and to do more through much smaller incision than those used by more traditional technique . it is a minimally invasive alternative for decompression of the ulnar nerve at the elbow , aiming to minimize the trauma to the tissues and improved postoperative recovery to the patients . its theoretical advantages over the classical open approach are the immediate well being of the patient , decreased invasiveness , minimal vascular complications , and less scar discomfort . in our view , like that patient with our inclusion criteria , 1992 , 1995 ; pavellza et al . , 2004 ; taniguchi et al , 2002 ; tsai et al . , 1999 ) is that the transposition of the ulnar nerve is not only unnecessary for the treatment of cubital tunnel syndrome , but that it may often be harmful and seriously disadvantageous , considering its potential complications ( heithofff , 1999 , mariani et al . , 1999 ) . the efficacy of simple decompression for the treatment of cubital tunnel syndrome was first reported by osborne ( 1957 ) . since then , many authors have reported good to excellent results with simple decompression ( chan et al . , 1980 ) . adelaar et al . , ( 1984 ) , bismmler and meyer ( 1996 ) , davies et al . , ( 1991 ) , and foster and edshage ( 1981 ) compared simple decompression with anterior transposition , and found no significant difference in clinical outcome . prospective randomized studies have shown results of simple decompression to be equal to those of anterior transposition [ 17 , 18 ] . in situ decompression also appears to have a low failure rate . but open decompression techniques require a longer incision , involve massive soft tissue dissection and are common associated with large postoperative scarring and wound tenderness . we can conclude that anterior transposition versus simple decompression has no significant difference in clinical outcome . a recent comparison between endoscopic techniques and in situ decompression demonstrated statistically significant less pain and greater satisfaction with the endoscopic technique . by minimally invasive with a direct visualisation to the ulnar nerve by endoscopic guidance can be visualised better more the potential compression sites of ulnar nerve entrapment , and all potential sites of nerve compression in the elbow region were released without damage to the macroscopically visible nerves . with endoscopy , a long portion of the nerve can be released without damage to cutaneous innervation . limited soft - tissue dissection with the preservation of the anatomy , especially vascularisation , minimises perineural fibrosis and enables rapid postoperative rehabilitation and can be safe and reliable with good functional and aesthetic result . according to the study by hoffmann and siemionow , the results of endoscopic release showed better functional recovery , lower morbidity and faster return to manual labour compared to conventional open method , and there were no serious complications . we conclude that endoscopic release is a safe procedure in the hands of the experienced surgeon with careful protection of the nerve and the branches and a complete decompression . the observed postoperative results demonstrated that this surgical technique to the ulnar nerve at the level of the elbow was very effective , and there was improvement in the clinical and electrophysiological outcomes in all the subjects who underwent the procedure . this procedure is a relatively alternative to the conventional open release technique in the uncomplicated cases . the short term has proven to be a safe and effective tool for the operative management of uncomplicated cases . the results showed better immediate functional recovery , lower morbidity , and faster and shorter rehabilitation time , and return to active activity was rapid or quicker return of the patients to their daily activity , acceptable aesthetic result and above all with a high patient satisfaction rate . endoscopic cubital tunnel release theoretically has better short - term outcome comparing to other technique in decompression but , however , to date , the number of studies reporting the case remains small . as part of a 629-patient , randomized , double - blind , multicenter clinical trial , 89 patients seen at the foothills medical center ( calgary , canada ) receive a 10-day course of vancomycin 125 mg 4 times daily or 200 mg twice daily of fidaxomicin orally as treatment of first episodes or first recurrences of cdi . patients submitted fecal samples , > 10 g / sample , at study entry ; on days 4 , 10 , 14 , 21 , and 28 ; and on day 3842 days . with the exception of specimens collected on weekends , which were refrigerated at 2c8c for processing on the following monday , 1-g aliquots of the samples were processed on the collection date for quantitative counts of c. difficile by serial dilution ( 10 , 10 , 10 , and 10 ) and plated onto cycloserine cefoxitin fructose agar ( ccfa ) , modified by using fastidious anaerobe agar base ( labm , bury , united kingdom ) . an additional 1-g aliquot was subjected to 100% ethyl alcohol shock ( 1:1 vol / vol 1-hour exposure ) and plated onto taurocholate - ccfa with the same dilution scheme as that used for total c. difficile counts . c. difficile cytotoxin b in fecal filtrates was measured by cell cytotoxicity assay , using vero cells with neutralization of cytotoxicity ( techlab , blacksburg , va ) . the toxin titer end point was the concentration showing 50% cell rounding ; titration range was 1/201/32 000 . to avoid confounding by concomitant antibiotic therapy for other indications , ten subjects who were in good health and using no antibiotics or medications donated fecal samples to serve as normal flora controls . fecal samples from 10 vancomycin- and 10 fidaxomicin - treated patients who had received no prior treatment for cdi before entry into the study , had sustained clinical cures of cdi ( ie , cure with recurrence ) and provided all samples to day 28 and variably to day 3842 were selected for microbiome characterization . the state of the microbiome was evaluated by quantitative real - time polymerase chain reaction ( qpcr ) , using previously published primers ( table 1 ) and methods [ 2025 ] . bacterial dna from 250 mg of fecal sample was extracted using the qiaamp dna stool mini kit ( qiagen , mississauga , canada ) . following the addition of lysis buffer and 200 mg of zirconium beads ( diameter , 0.2 mm ) , purified genomic dna ( gdna ) was eluted off the columns with 200 l of molecular - grade water . the bacterial gdna was checked for purity and concentration , using the nanodrop 2000 spectrophotometer ( thermo scientific , wilmington , de ) . yield ( in nanograms of dna per milligram of stool ) was calculated in samples , after which they were stored at 20c for later analysis . qpcr was performed using iq5 and cfx96 detection systems ( biorad , mississauga , canada ) . amplification and detection were conducted in 96-well plates with sybr green 2 qpcr master mix ( biorad ) . each sample was run in duplicate in a final volume of 20 l containing a final concentration of 0.3 m of each primer and 5 l of 4-ng/l template gdna . amplifications were conducted using the 2-step template ramping profile : 1 cycle at 95c for 5 minutes , followed by 49 cycles at 95c for 30 seconds , 52c60c for 30 seconds , and 72c for 30 seconds . table 1.16s ribosomal rna probes used for quantitative real - time polymerase chain reaction to quantify shifts in major components of the fecal flora during and after treatment of clostridium difficile infectiongroupnamesequencereferencebacteroidesbac303fgaaggtcccccacattgbernhard et al , 2000bacteroidesbac708rcaatcggagttcttcgtgbernhard et al , 2000enterobacteriaceaeeco1457fcattgacgttacccgcagaagaagcbartosch et al , 2004enterobacteriaceaeeco1652ctctacgagactcaagcttgcbartosch et al , 2004clostridium leptum subgroupsg - clept - fgcacaagcagtggagtmatsuki et al , 2002c . difficileg - cdif - rccatcctgtactggctcacctrinttil et al , 2004desulfovibrio desulfuricans groupg - desulf - fggtaccttcaaaggaagcacrinttil et al , 2004d . desulfuricans groupg - desulf - rgggatttcacccctgacttarinttil et al , 2004enterococcus speciesg - enter - fcccttattgttagttgccatcattrinttil et al , 2004enterococcus speciesg - enter - ractcgttgtacttcccattgtrinttil et al , 2004veillonella speciesg - veill - fa(c / t)caacctgcccttcagarinttil et al , 2004veillonella speciesg - veill - rcgtcccgattaacagagcttrinttil et al , 2004prevotellacfb286fgtaggggttctgagaggaprobebase pb-00045prevotellacfb719ragctgccttcgcaatcggprobebase pb-00047lactobacillusuni331ftcctacgggaggcagcagtnadkarni et al , 2002lactobacilluslacto371rtgg aag att ccc tac tgcprobebase pb-00195bifidobacterium speciesbif551fcgcgtcyggtgtgaaagdelroisse et al , 2008bifidobacterium speciesbif794rccccacatccagcatccadelroisse et al , 2008 16s ribosomal rna probes used for quantitative real - time polymerase chain reaction to quantify shifts in major components of the fecal flora during and after treatment of clostridium difficile infection the specificity of the primers was determined by using purified template dna from reference strains , including bacteroides fragilis american type culture collection ( atcc ) 25825 , bacteroides thetaiotaomicron atcc 29741 , bacteroides distasonis atcc 8503 , bacteroides ovatus atcc 8483 , bacteroides vulgatus atcc 8482 , prevotella melaninogenica atcc 25845 , clostridium coccoides atcc 29236 , fusobacterium nucleatum subspecies nucleatum atcc 25586 , peptostreptococcus anaerobius atcc 27337 , bifidobacterium bifidum atcc15696 , and c. difficile atcc 43255 . melting curve analysis was performed to ensure the specificity of the primer sets and subsequent qpcr amplification reactions . the limit of detection for each assay was determined with concentrations of purified dna of the reference strains ranging from 0.0000220 ng . this range was also used to generate each standard curve using serial 10-fold dilutions . to estimate the amounts of target bacterial dna from fecal dna extractions , threshold cycle ( ct ) values were first converted into gdna copies using the standard curve generated from each qpcr run . for construction of standard curves , 10-fold - dilution series of 1010 target genomes from target species were prepared and run with samples . to calculate colony - forming units ( cfu ) per gram of stool , the target genomes calculated from 20 ng of gdna ( the amount used per qpcr reaction ) were extrapolated into cfu per gram of stool by multiplying the cfu per nanogram of gdna by the yield ( total nanograms of gdna extracted from 1 g of stool ) . the majority of the qpcr tests were designed to detect a wide range of related and diverse bacterial species that , most likely , did not have the same genome size or ribosomal dna copy numbers . therefore in addition to the foregoing , 5 normal control samples were analyzed pair wise by quantitative cultures and by qpcr for bacteroides group organisms , enterobacteriaceae , and enterococcus species . the quantitative bacterial counts of c. difficile and calculated cfu per gram of target organism group by real - time qpcr were log transformed , after which changes in each time of collection were compared between fidaxomicin and vancomycin treatments , using wilcoxon signed - rank tests for nonparametric data ( graphpad prism ; graphpad , san diego , ca ) . the specificity of the primers was determined by using purified template dna from reference strains , including bacteroides fragilis american type culture collection ( atcc ) 25825 , bacteroides thetaiotaomicron atcc 29741 , bacteroides distasonis atcc 8503 , bacteroides ovatus atcc 8483 , bacteroides vulgatus atcc 8482 , prevotella melaninogenica atcc 25845 , clostridium coccoides atcc 29236 , fusobacterium nucleatum subspecies nucleatum atcc 25586 , peptostreptococcus anaerobius atcc 27337 , bifidobacterium bifidum atcc15696 , and c. difficile atcc 43255 . melting curve analysis was performed to ensure the specificity of the primer sets and subsequent qpcr amplification reactions . the limit of detection for each assay was determined with concentrations of purified dna of the reference strains ranging from 0.0000220 ng . to estimate the amounts of target bacterial dna from fecal dna extractions , threshold cycle ( ct ) values were first converted into gdna copies using the standard curve generated from each qpcr run . for construction of standard curves , 10-fold - dilution series of 1010 target genomes from target species were prepared and run with samples . to calculate colony - forming units ( cfu ) per gram of stool , the target genomes calculated from 20 ng of gdna ( the amount used per qpcr reaction ) were extrapolated into cfu per gram of stool by multiplying the cfu per nanogram of gdna by the yield ( total nanograms of gdna extracted from 1 g of stool ) . the majority of the qpcr tests were designed to detect a wide range of related and diverse bacterial species that , most likely , did not have the same genome size or ribosomal dna copy numbers . in addition to the foregoing , 5 normal control samples were analyzed pair wise by quantitative cultures and by qpcr for bacteroides group organisms , enterobacteriaceae , and enterococcus species . the quantitative bacterial counts of c. difficile and calculated cfu per gram of target organism group by real - time qpcr were log transformed , after which changes in each time of collection were compared between fidaxomicin and vancomycin treatments , using wilcoxon signed - rank tests for nonparametric data ( graphpad prism ; graphpad , san diego , ca ) . of the 89 patients randomly assigned in the study , 44 vancomycin- and 45 fidaxomicin - treated patients responded to treatment . c. difficile strain typing showed that 13% of strains were ribotype 027 . recurrence was observed in 23% of vancomycin - treated patients ( 10 of 44 ) and in 11% of fidaxomicin - treated patients ( 5 of 44 ) . results of qpcr analysis of fecal samples obtained from healthy donors are summarized in table 2 . paired tests showed that qpcr results were approximately 1 log10 cfu / g lower than results by quantitative culture . table 2.results of quantitative real - time polymerase chain reaction detection of components of the microflora from 10 healthy control donors of stoolmean log10 cfu / g sdmean log10 cfu / g sdbacteroides9.66 0.28bifidobacterium6.78 0.86prevotella9.20 0.50veillonella5.80 0.90clostridium coccoides9.02 0.53desulfovibrio5.28 1.24clostridium leptum7.73 0.77enterobacteriaceae5.53 0.75lactobacillus7.59 0.51enterococcus species5.30 0.75abbreviations : cfu , colony - forming unit ; sd , standard deviation . results of quantitative real - time polymerase chain reaction detection of components of the microflora from 10 healthy control donors of stool abbreviations : cfu , colony - forming unit ; sd , standard deviation . figures 110 show counts of target organisms in individual patients over time after receipt of vancomycin or fidaxomicin , as well as mean values for 10 healthy controls . clinical trial site subject numbers have been removed , and subjects are listed sequentially by number to preserve confidentiality . mean counts of bacteroides species , prevotella species , c. coccoides , and clostridium leptum were approximately 2 log counts lower at study entry , compared with results from healthy control fecal samples . for samples collected on day 4 , a washout effect was noted on the basis of a transitory dip in cfu - per - gram for fidaxomicin - treated subjects , likely as a result of continued diarrhea , whereas the decrease in microbial counts was substantially greater for vancomycin - treated subjects , presumably because of both a washout effect plus microbial suppression / killing . as a result , the differences between the day-0 and day-10 samples at the end of treatment appear to be the best comparators of the effect of treatment on microbial counts . vancomycin markedly suppressed bacteroides , prevotella , c. coccoides , and c. leptum group organisms and suppressed bifidobacteria species to a lesser extent . fidaxomicin appeared to spare ( for most groups ) or act indifferently ( for bifidobacteria species ) on these groups . veillonella species counts increased during vancomycin treatment and decreased in the post - treatment phase , whereas counts were unchanged in fidaxomicin - treated patients , suggesting an opportunistic increase in counts during suppression of other organism groups during vancomycin treatment . vancomycin suppressed counts of non vancomycin - resistant enterococci during therapy , after which there was a rebound in counts and gradual reduction in numbers later in the follow - up period . counts of enterobacteriaceae were 23 logs higher than those in healthy controls at study entry and , for both treatments , appeared to drift downward in the posttreatment period . figure 1.bacteroides microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 2.clostridium coccoides microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . abbreviation : cfu , colony - forming units . figure 3.clostridium leptum microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 4.prevotella microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 5.veillonella microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 6.bifidobacterium microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 7.lactobacillus microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 8.desulfovibrio microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 9.enterobacteriaceae microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . figure 10.enterococcaceae microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . bacteroides microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . clostridium coccoides microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . clostridium leptum microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . prevotella microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . veillonella microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . abbreviation : cfu , colony - forming units . bifidobacterium microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . lactobacillus microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . desulfovibrio microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . enterobacteriaceae microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . enterococcaceae microflora levels over time in 10 patients receiving vancomycin ( a ) , 10 patients receiving fidaxomicin ( b ) , and 10 healthy controls ( boxes ) . abbreviation : cfu , colony - forming units . figure 11a c and table 3 summarize the effect of vancomycin versus that of fidaxomicin during and following cdi treatment for 20 patients who achieved sustained cure . the day 3842 samples , which were collected in 34 patients in each group , made comparisons difficult at the end of the follow - up period . because most recurrences in the trial occurred within 23 weeks of completing therapy , the comparison of samples obtained at study entry to day-28 samples likely illustrates the differences between treatments . at study entry , for bacteroides / prevotella organisms , vancomycin markedly suppressed this organism group during therapy . even at day 28 , significant differences were noted between vancomycin and fidaxomicin . similar differences were observed for the c. coccoides and c. leptum groups , but suppression by vancomycin was transient , with recovery by day 21 . figure 11.levels of major cultivable and noncultivable genera of the normal fecal microbiota in 20 patients with clostridium difficile infection who were randomly assigned to receive treatment with fidaxomicin or vancomycin ( 10 patients / treatment arm ) . table 3.results of quantitative real - time polymerase chain reaction for major bacterial groups for 10 patients receiving vancomycin or fidaxomicin for 10 days , followed by 4 weeks of follow - upbacteroidesprevotellaclostridium coccoides groupclostridium leptum groupbifidobacteriaenterobacteriaceaelactobacillusenterococcaceaemean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sdmean log10 cfu sddayvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicinvancomycinfidaxomicin07.61 2.218.52 1.537.62 2.178.11 1.667.89 1.588.17 0.715.41 2.215.81 2.226.96 0.726.35 1.427.50 1.707.94 0.707.55 0.627.62 0.546.17 0.515.48 1.3544.26 0.887.81 1.545.30 0.817.84 1.614.73 0.536.91 0.942.04 0.393.53 5.085.30 0.815.30 1.338.52 1.468.81 0.877.90 0.457.10 0.785.23 0.315.75 1.18105.26 0.919.33 1.305.50 0.928.84 1.194.68 0.738.49 0.552.02 1.315.08 2.265.17 1.096.28 1.408.84 0.577.84 1.187.91 0.707.64 0.655.43 0.365.93 0.95145.99 2.109.52 1.195.43 1.309.06 1.046.74 2.218.65 0.444.03 1.835.29 2.376.56 1.436.18 1.758.01 1.047.98 0.808.11 0.437.66 0.736.26 0.856.07 1.22215.95 2.309.30 1.446.63 2.218.97 1.378.51 0.648.73 0.585.85 1.215.83 2.196.94 0.936.06 1.697.62 1.037.06 0.927.69 0.487.34 0.936.15 0.676.12 0.90286.95 2.879.08 1.166.83 2.548.93 0.858.66 1.118.07 1.456.18 1.015.82 1.355.89 1.196.10 1.357.33 1.185.95 2.147.65 0.557.23 1.095.77 0.725.79 0.73427.83 1.709.34 0.737.89 1.639.22 0.358.41 0.698.40 0.827.09 1.336.89 1.085.62 1.516.11 1.176.52 1.616.50 1.107.50 0.507.60 0.805.77 0.765.71 0.79data are cfu per gram of stool . results support data in figures 110.abbreviations : cfu , colony - forming unit ; sd , standard deviation . levels of major cultivable and noncultivable genera of the normal fecal microbiota in 20 patients with clostridium difficile infection who were randomly assigned to receive treatment with fidaxomicin or vancomycin ( 10 patients / treatment arm ) . results of quantitative real - time polymerase chain reaction for major bacterial groups for 10 patients receiving vancomycin or fidaxomicin for 10 days , followed by 4 weeks of follow - up data are cfu per gram of stool . table 4 summarizes the results of total quantitative cultures for c. difficile at study entry , day 4 , day 10 , and in the follow - up period , along with spore counts and c. difficile cytotoxin b titers . mean total and spore counts at study entry were similar between treatment arms . at days 4 and 10 , both treatments effectively reduced c. difficile counts to the lower limit of quantitation , after which there appeared to be a similar low - level rebound of c. difficile counts . toxin titers were similar at study entry , and all patients had no detectable toxin ( titer , < 1/20 ) on days 410 of treatment . however , vancomycin - treated patients were twice as likely to reexpress toxin in the follow - up period ( p = .03 ) , primarily within the first 2 weeks of treatment . in the total patient group of 89 patients , patients who achieved sustained cure , vancomycin - treated patients who experienced recurrence of cdi had significantly lower counts of bacteroides and prevotella organisms at the end of treatment ( table 5 ) . a trend toward lower counts of these organisms was also observed in fidaxomicin - treated patients who subsequently experienced recurrence of cdi . at diagnosis of recurrent cdi , toxin titers , pathogen counts , and microbiome profiles were similar to findings at study entry ( data not shown ) . table 4.quantitative cultures for clostridium difficile total counts , spore post alcohol shock counts , and c. difficile cytotoxin b titers in 89 patients randomly assigned to receive a 10-day course of vancomycin or fidaxomicin therapyday 0day 4day 10day 14day 21day 28day 42mean log10 cfu / g sd ( spore sd ) van6.2 2.12.2 0.92.0 0.03.1 2.24.5 2.73.9 2.24.5 2.4 fdx5.9 2.82.0 0.12.0 0.12.7 1.94.2 2.43.9 2.33.0 1.9mean log10 spore count ( sd ) van4.8 1.72.7 1.12.2 0.63.2 2.04.0 1.93.3 1.53.2 1.6 fdx4.7 1.82.0 0.02.1 0.72.3 1.23.3 1.83.1 1.42.7 1.3mean toxin b titer semday 0day 4day 10pooled for days 14 , 21 , 28 , 42 van2800 1250negativenegative1260 350 fdx2250 600negativenegative2400 1400a total of 44 patients received van , and 45 received fdx.abbreviations : cfu , colony forming units ; fdx , fidaxomicin ; sd , standard deviation ; sem , standard error of the mean ; van , vancomycin . several values were higher than total counts , reflecting several samples for which spore counts appeared higher than total counts , which could represent variation near the lower limit of detection and possibly less competition after alcohol shock treatment cultures . data are for positive titers only . for the van group , 7 of 30 specimens tested from day 14 , 15 of 30 from day 21 , 3 of 22 from day 28 , and 1 of 12 from day 42 were positive . for the fdx group , 1 of 23 specimens tested from day 14 , 6 of 27 from day 21 , 5 of 20 from day 28 , and 1 of 21 from day 42 were positive . the value was 1140 770 if the outlier toxin titer of 1/16 000 was excluded . table 5.comparison of changes in microflora among patients who achieved sustained cure of clostridium difficile infection with changes among patients who achieved cure but had subsequent recurrencelog10 cfu / g feces sdpatients with sustained curepatients with recurrenceday 0day 10day 0day 10bacteroides group vancomycin7.60 2.215.26 0.918.40 2.014.37 0.39 fidaxomicin8.52 1.539.33 1.305.76 1.947.00 2.33prevotella species vancomycin7.62 2.175.50 0.928.11 1.994.43 0.67 fidaxomicin8.11 1.668.84 1.195.26 1.966.57 2.20clostridium coccoides group vancomycin7.89 1.584.67 0.728.46 0.514.95 1.30 fidaxomicin8.17 0.718.49 0.557.21 1.776.80 2.77ten patients in the vancomycin group and 10 in the fidaxomicin achieved sustained cure , whereas 10 in the vancomycin group and 5 in the fidaxomicin group had recurrence . fidaxomicin - treated patients with recurrence appeared to have greater reductions in bacteroides counts at study entry.abbreviation : cfu , colony - forming unit . p < .03 , compared with day 10 values for patients with recurrence . p = .03 , compared with day 10 values for patients with recurrence . p = .08 , compared with day 10 values for patients with recurrence . p = .007 , compared with day 10 values for patients with recurrence . p = .11 , compared with day 10 values for patients with recurrence . quantitative cultures for clostridium difficile total counts , spore post alcohol shock counts , and c. difficile cytotoxin b titers in 89 patients randomly assigned to receive a 10-day course of vancomycin or fidaxomicin therapy a total of 44 patients received van , and 45 received fdx . abbreviations : cfu , colony forming units ; fdx , fidaxomicin ; sd , standard deviation ; sem , standard error of the mean ; van , vancomycin . several values were higher than total counts , reflecting several samples for which spore counts appeared higher than total counts , which could represent variation near the lower limit of detection and possibly less competition after alcohol shock treatment cultures . 7 of 30 specimens tested from day 14 , 15 of 30 from day 21 , 3 of 22 from day 28 , and 1 of 12 from day 42 were positive . for the fdx group , 1 of 23 specimens tested from day 14 , 6 of 27 from day 21 , 5 of 20 from day 28 , and 1 of 21 from day 42 were positive . the value was 1140 770 if the outlier toxin titer of 1/16 000 was excluded . comparison of changes in microflora among patients who achieved sustained cure of clostridium difficile infection with changes among patients who achieved cure but had subsequent recurrence ten patients in the vancomycin group and 10 in the fidaxomicin achieved sustained cure , whereas 10 in the vancomycin group and 5 in the fidaxomicin group had recurrence . fidaxomicin - treated patients with recurrence appeared to have greater reductions in bacteroides counts at study entry . abbreviation : cfu , colony - forming unit . p < .03 , compared with day 10 values for patients with recurrence . p = .03 , compared with day 10 values for patients with recurrence . p = .08 , compared with day 10 values for patients with recurrence . p = .007 , compared with day 10 values for patients with recurrence . p = .11 , compared with day 10 values for patients with recurrence . cdi is the most prominent clinical example of the consequence of impairment of the normal microbiota of the gut , resulting in loss of colonization resistance , pathogen proliferation , and diarrheal disease . measurement of the degree of impairment of the intestinal flora that is thought to occur with intestinal infections has awaited new technologies available only in the past decade . studies to date have shown depletion of bacteroides species and firmicutes in several patients experiencing multiple recurrences , floral shifts by denaturing gradient gel electrophoresis , and changes in bacteroidetes and firmicutes , as determined by microarray signaling , in a comparison of cohorts of patients with and control patients without cdi . a systematic prospective and quantitative study to evaluate microbiome shifts during fidaxomicin treatment of cdi is reported here . the extent and nature of impairment of the microbiome before the onset of cdi diarrheal symptoms ( equivalent to the concept of the latent period ) are unknown . fecal samples could also be regarded as an indirect measure of biological processes because events on the mucosal interface likely are more determinative in pathogenesis . nevertheless , given the technical challenges of obtaining and separating microbes in the fecal stream from those on surfaces , comparison of microbiome profiles in fecal samples from healthy controls with samples at the onset of cdi are a measure of the degree of impairment of the microbiome . findings in this study extend and support results of the phase ii clinical study that used conventional culture and fluorescence in situ hybridization . bacteroides and firmicutes groups ( c. coccoides plus c. leptum ) , which constitute the bulk of the microflora , appear to be reduced 10100-fold at the time of cdi diagnosis on the basis of qpcr testing . on the basis of paired culture versus qpcr results , using bacteroides as an indicator group , the mean reduction could be 1-log10 greater . there is substantial variation in the degree of suppression of the normal microbiota , with some patients showing bacteroides counts in the 10-cfu / g range . because diarrheal movements could have a washout effect on normal flora , it is likely that a decreased degree of perturbation of the microbiome can result in cdi . because the decrease in counts at day 4 appears to be a further washout effect due to diarrheal movements before disease resolution ( with or without antimicrobial suppression ) , differences in counts at study entry and at the end of treatment at day 10 have been used to quantify the effect of drug treatment on the residual microbiome . the results of microbiome profiling document the collateral damage to the microflora seen with vancomycin therapy , a further 23-log decrease in bacteroides group counts resulting in a 46-log decrease from healthy control counts at the end of vancomycin therapy . the observation that bacteroides organisms are more numerous on the mucosal surface compared with the feces itself suggests an important role in colonization resistance , perhaps by a process of bacterial interference versus intestinal pathogens . this decrease is associated with reappearance of c. difficile cytotoxin b in fecal filtrates at a frequency that is twice that after fidaxomicin therapy and , in this study population , was accompanied by twice the number of cdi recurrences . these findings are consistent with outcomes in the clinical trials as a whole [ 11 , 12 ] and underscore the important observation that the majority of recurrences eventuate during the 23-week interval after treatment . a reduction ( p = .001 ) in number of spores at day 4 of fidaxomicin treatment , compared with day 4 of vancomycin treatment ( table 4 ) , the c. difficile microbial profiles were not different between treatments and therefore support the alternate hypothesis that reduced recurrence is related to the health of the residual normal microflora . comparison of the pattern of suppression of members of the microbiome between the 2 treatments suggests that bacteroides species , prevotella species , the firmicutes , and bifidobacteria species play a beneficial role in maintaining a healthy microbiome and that high counts of coliforms and enterococci are a marker for a depleted gut microbiome . the observation that patients with recurrence of cdi have lower counts of bacteroides and prevotella organisms at the end of therapy is additional supportive evidence of a protective role of these microbes . in the final analysis , the unchanged counts seen in this study neither support nor refute a role for lactobacilli in preventing cdi recurrences . on the basis of the high efficacy of fecal microbiome transplantation in arresting recurrent cdi , it is likely that the dominant members of the microbiome are critical for preventing cdi . in summary , the microflora - sparing features of fidaxomicin shown in this report offer selective therapy for cdi , a more robust microbiome , and a decreased risk for recurrent disease .

cubital tunnel syndrome is one of the common upper extremity problem encountered . a mild syndrome can be often treated without surgery , but a failure of conservative treatment with constant symptoms or muscle atrophy and weakness requires surgical intervention . despite the fact that is the second most common nerve entrapment in the upper limb , there is no accepted gold standard in the surgical management . but with the new technique in minimally invasive surgery and available endoscope , it addresses all potential compression sites with good visualisation but with small surgical exposure . the procedure is safe and reliable way to address this problem . the microflora - sparing properties of fidaxomicin were examined during the conduct of a randomized clinical trial comparing vancomycin 125 mg 4 times per day versus fidaxomicin 200 mg twice per day for 10 days as treatment of clostridium difficile infection ( cdi ) . fecal samples were obtained from 89 patients ( 45 received fidaxomicin , and 44 received vancomycin ) at study entry and on days 4 , 10 , 14 , 21 , 28 , and 38 for quantitative cultures for c. difficile and cytotoxin b fecal filtrate concentrations . additionally , samples from 10 patients , each receiving vancomycin or fidaxomicin , and 10 samples from healthy controls were analyzed by quantitative real - time polymerase chain reaction with multiple group - specific primers to evaluate the impact of antibiotic treatment on the microbiome . compared with controls , patients with cdi at study entry had counts of major microbiome components that were 23-log10 colony - forming units ( cfu)/g lower . in patients with cdi , fidaxomicin allowed the major components to persist , whereas vancomycin was associated with a further 24-log10 cfu reduction of bacteroides / prevotella group organisms , which persisted to day 28 of the study , and shorter term and temporary suppression of both clostridium coccoides and clostridium leptum group organisms . in the posttreatment period , c. difficile counts similarly persisted in both study populations , but reappearance of toxin in fecal filtrates was observed in 28% of vancomycin - treated patient samples ( 29 of 94 ) , compared with 14% of fidaxomicin - treated patient samples ( 13 of 91 ; p = .03 ) . similarly , 23% of vancomycin - treated patients ( 10 of 44 ) and 11% of fidaxomicin - treated patients ( 5 of 44 ) had recurrence of cdi . whereas vancomycin and fidaxomicin are equally effective in resolving cdi symptoms , preservation of the microflora by fidaxomicin is associated with a lower likelihood of cdi recurrence.clinical trials registration . ntc00314951 .

magnetic resonance imaging ( mri ) is a powerful noninvasive imaging technique , providing high - resolution anatomical images with excellent soft - tissue contrast . a large fraction of mri studies utilize a contrast agent , e.g. , more than half the clinical studies performed at our institution make use of a gadolinium(iii)-based contrast agent . these contrast agents are simple , water - soluble , ternary gadolinium(iii ) complexes with gadolinium(iii ) coordinated by an octadentate poly(aminocarboxylato ) ligand and an aqua coligand . gadolinium(iii ) is advantageous for use in mri contrast agents because of its large electron spin number ( s = /2 ) and long electronic relaxation time , which result in efficient nuclear relaxation of nearby solvent water molecules . contrast agents are characterized by their relaxivity , defined as the change in the solvent relaxation rate ( 1/t1 or 1/t2 ) normalized to the concentration of the contrast agent ( units of mm s ) . first - generation clinical contrast agents are monomeric gadolinium(iii ) complexes based on a [ gd(dota)(h2o ) ] or [ gd(dtpa)(h2o ) ] ( dtpa = diethylenetriaminepentaacetic acid ) framework and have relaxivities in the range 35 mm s. second - generation contrast agents such as gadofosveset ( ms-325 , ablavar ) used reversible protein binding to increase the relaxivity . protein binding slows the rotational tumbling rate of the complex , and as this rate approaches the proton larmor frequency , nuclear relaxation is enhanced . specific protein binding also serves to direct the contrast agent to specific pathologies , e.g. , thrombosis , as well as enhance the relaxivity . however , the increase in the relaxivity due to protein binding is greatest by far at low magnetic fields ( 1.5 t ) and falls off precipitously at higher fields . clinically , 3 t mri is becoming common , and the major equipment vendors all sell 7 t scanners . for small - animal imaging , even higher field strengths are common . at high fields ( 3 t ) , the proton larmor frequency is much faster than the tumbling rate of protein - bound complexes but still much slower than the tumbling rate of discrete monomeric complexes . we and others have reported multimeric contrast agents with increased high - field relaxivity due to a tumbling rate intermediate between that of protein - bound agents and small molecules . multiple gd(dotala ) moieties could be incorporated into oligopeptides of defined structure and size , resulting in relaxivities tuned for high - field applications . the modular nature of gd(dotala ) also lends itself to the design of serum albumin - targeted contrast agents . here , we report on the synthesis and properties of a modified derivative of the dotala ligand , dotalap . we sought to improve the properties of the dotala ligand by replacing one of the acetate moieties by methylenephosphonate . the increased charge of the phosphonate is expected to increase the solubility of the resultant gadolinium(iii ) complexes . gadolinium(iii ) complexes with phosphonate - containing ligands often yield higher relaxivities than complexes of the analogous carboxylate ligand due to an effect on water in the second coordination sphere , and this second - sphere effect persists at high fields . we also expected that inner - sphere water exchange should be faster for the phosphonate - substituted ligand . for gadolinium(iii ) complexes undergoing water exchange via a dissociative mechanism , substitution of a carboxylate with the bulkier phosphonate donor group results in faster water exchange as the energy difference between the 8-coordinate transition state and the 9-coordinate ground state becomes smaller . on the other hand , this steric crowding can also result in an 8-coordinate ground state with an inner - sphere water ligand eliminated , which would result in lower relaxivity . in this study , we sought to address the following questions : ( i ) how does substitution of one of the acetate donors in dotala for methylenephosphonate impact the hydration number and water exchange kinetics and , in turn , the relaxivity ? ( ii ) how does phosphonate substitution affect kinetic inertness with respect to gadolinium(iii ) dissociation ? ( iii ) what is the effect of amine versus carboxylate functionalization of the propionamine arm of dotalap on the hydration number , water exchange kinetics , relaxivity , and gadolinium(iii ) dissociation kinetics ? ( iv ) are any of these properties altered when a gd(dotalap ) derivative is bound to serum albumin ? ( v ) does phosphonate substitution confer enhanced high - field relaxivity properties and , if so , do these result in an increased in vivo mri signal ? to address these questions , we synthesized the dotalap ligand by altering the previously established dotala synthesis . additionally , we prepared four dotalap derivatives substituted at either the amine or carboxylate of the propionamine arm . the gadolinium(iii ) complexes were prepared and characterized with respect to variable - field , variable - temperature relaxivity , water exchange kinetics , albumin binding studies , and gadolinium(iii ) dissociation kinetics . europium(iii ) complexes were also synthesized , and luminescence lifetime measurements were performed to quantify the hydration number of these complexes . benzyl 2-[[(benzyloxy)carbonyl]amino]-3-(1,4,7,10-tetraazacyclododecan-1-yl)propanoate represents an intermediate common to both dotala and dotalap , so we used the previously established synthetic steps to furnish this first intermediate ( scheme 1 ) . in order to introduce a protected phophonate arm onto the monoderivatized cyclen macrocycle , tri - tert - butyl phosphite was synthesized according to ref ( 22 ) and appended using a mannich - type reaction . the reaction was monitored for disappearance of the monofunctionalized macrocycle using liquid chromatography mass spectrometry ( lc ms ) , followed by filtration and aqueous workup of the filtrate . the introduction of these two chelator arms at this stage is key : because of their steric hindrance , it is not possible to incorporate them efficiently at a later stage . the crude intermediate containing the difunctionalized macrocycle was then redissolved in acetonitrile and subjected to alkylation with tert - butyl bromoacetate , followed by preparative high - performance liquid chromatography ( hplc ) to purify the protected intermediate compound 1 . orthogonal protection group chemistry provided us with a free and ready - to - couple carboxylate and primary amine on compound 2 after hydrogenation - mediated removal of the carboxybenzyl ( cbz ) and benzyl protecting groups of 1 . the overall number of steps starting from cbz - nh - ser - oh - obn is 5 , and compound 2 is obtained with an approximate 10% overall yield based on the protected serine . the lower yield is due to the formation of a greater number of side products compared to the dotala synthesis . one single phosphonate species ( 1,7-functionalized with respect to the aminopropanoate arm ) is obtained and isolated , as indicated to us by h nmr . compound 2 also represents the last common intermediate for the synthesis of compounds 35b , which all are furnished by activation of the carboxylate with 1-[bis(dimethylamino)methylene]-1h-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate ( hatu ) in the presence of diisopropylethylamine ( dipea ) as a base , followed by overnight amide coupling and subsequent hplc purification . the coupled , purified products still carry tert - butyl ester protective groups on the acetate groups and the phosphonate . these esters are then removed using an acid - catalyzed deprotection reaction with trifluoroacetic acid in dichloromethane , yielding the final ligands without the formation of side products . complexes were formed under standard conditions by mixing the ligand with an aqueous solution of the lanthanide trichloride salt . the ph of the solution was adjusted to 7 by the dropwise addition of a 0.1 m solution of naoh . complexation was followed by hplc in order to confirm that > 95% of ligand was complexed . we also used the xylenol orange test to further confirm that no free lanthanide was present . according to our hypothesis , gd(dotalap ) derivatives should display water exchange rates that exceed the previously reported water exchange rates of gd(dotala ) derivatives . we determined the water exchange rates of the gadolinium(iii ) complexes by variable - temperature measurement of the transverse relaxation time of h2o in the presence and absence of each gadolinium(iii ) complex at 11.7 t. in the case of gd(4a ) , gd(4b ) , and gd(5b ) , we observed only minor changes in the transverse relaxation rate when the complexes were added , indicating a lack of an inner - sphere water ligand for these complexes . for instance , the maximum o relaxivity , r2 , was 2.5 mm s for these three complexes , while the maximum r2 is typically > 15 mm s for q = 1 complexes at this field strength . on the other hand , gd(3 ) and gd(5a ) had much higher maximum r2 values consistent with an inner - sphere water ligand ( 18 and 16 mm s , respectively ) . figure 1 shows the reduced transverse relaxation rate r2r as a function of the reciprocal temperature . the crossover to exchange - limited r2r occurs at a low temperature for both complexes , which indicates the predicted , very fast water exchange kinetics . we used a four - parameter model described previously to fit the data and obtained water residency times at 37 c of m = 8.1 ns for gd(3 ) and m = 6.4 ns for gd(5a ) ; see table 1 . while these water residency times are considerably shorter than those seen for [ gd(dota)(h2o ) ] and [ gd(dotala)(h2o ) ] , they are similar to the water residency time for [ gd(do3ap)(h2o ) ] , a close structural analogue of dotalap ( table 1 ) . the coordination spheres of gd(3 ) and gd(5a ) differ in that gd(3 ) contains an anionic propionate oxygen donor and gd(5a ) contains a neutral propionamide oxygen donor but their water exchange kinetics are similar . the propionate to propionamide substitution does not result in different water exchange kinetics in other gadolinium(iii ) complexes . this is in contrast to the acetate to acetamide substitution , which is well established to reduce the water exchange rate . interestingly , substitution at the propionamine [ gd(4a ) and gd(4b ) ] gives a ligand with the same donor set as 3 , but these peripheral modifications now result in a gadolinium complex with no inner - sphere water ligand . similarly , changing the amide substituent from gd(5a ) to gd(5b ) results in the latter complex having no inner - sphere water ligand . for complexes with exceedingly fast water exchange kinetics , these results indicate that seemingly minor modifications to the periphery of the ligand can alter the hydration number of the complex . temperature dependence of the o nmr ( 11.7 t ) reduced transverse relaxation rates of gd(3 ) ( 6.73 mm , left ) and gd(5a ) in pbs ( 4.16 mm , right ) . the solid line represents a fit to the data to determine the mean water residency time m . determination of the relaxivity of the gadolinium(iii ) complexes also gives further indication of their hydration state . the longitudinal proton relaxivity ( r1 ) was measured in ph 7.4 pbs at 37 c at two larmor frequencies , 20 and 60 mhz . for small molecules with q > 0 , the relaxivity is dominated by the short rotational correlation time at these frequencies and the hydration state . additionally , phosphonate - containing gadolinium(iii ) chelates have been reported to result in increased second - sphere relaxivity . if all of the complexes were q = 1 , then we would expect the relaxivity to increase proportionally with the molecular weight , ranking them as follows : gd(3 ) < gd(4a ) gd(5a ) < gd(4b ) gd(5b ) . in fact , only gd(3 ) and gd(5a ) behave as expected with a relatively high , but for q = 1 phosphonato complexes typical , relaxivity . on the other hand , gd(4a ) , gd(4b ) , and gd(5b ) show relaxivities below 4 mm s , which is more typical for q = 0 complexes with elevated outer - sphere relaxivity ( table 2 ) . these relaxivity measurements are consistent with the o nmr measurements , which suggests the lack of an inner - sphere water ligand for gd(4a ) , gd(4b ) , and gd(5b ) . the hydration number of a gadolinium(iii ) complex can be estimated using a surrogate complex with the luminescent lanthanide ions eu or tb . this is done by measuring the rate constants for luminescence decay in d2o and h2o solutions . an empirical expression originally reported by horrocks and sudnick relates these rate constants to q ( table 3 ) . it is then assumed that gadolinium(iii ) has the same hydration number based on the similar ionic radii of these ions and their propensity for forming isostructural complexes . however , application of this method to phosphonate - containing complexes typically results in an overestimation of q by 0.30.6 . with this consideration in mind , we find that the q values obtained mirror the relaxometric behavior of the corresponding gadolinium complexes . the hydration numbers calculated are proportional to the obtained relaxivity values in pbs at 20 and 60 mhz , with eu(3 ) and eu(5a ) providing q values above 1 , while the other complexes show lower q values . figure s1 ( supporting information ) shows the correlation of the 20 mhz relaxivity values of the gadolinium(iii ) complex and the apparent hydration number of the corresponding europium(iii ) complex . nd = not determined . because of the decreased solubility of eu(5b ) compared to the other europium complexes described here , we were not able to generate a solution with high enough complex concentration required for this measurement . we had previously conjugated ibuprofen to dotala and found that a 0.1 mm solution of its gadolinium(iii ) complex [ compound gd(6 ) , figure 2 ] was 70% bound to 4.5% ( w / v ) hsa , and this complex also displayed high relaxivity ( at low fields ) in the presence of hsa . to understand how the phosphonate modification affects protein binding and relaxivity for a slow tumbling system , we synthesized the ibuprofen conjugates gd(4b ) and gd(5b ) shown in figure 2 . structures of ibuprofen conjugates gd(4b ) and gd(5b ) reported here , as well as previously reported compound gd(6 ) . compared to gd(6 ) , the affinity of gd(4b ) for hsa was increased 3-fold ( kd = 260 and 80 m , respectively ) . the reason for the higher percentage of bound complex in the case of gd(4b ) is likely the double negative charge , which may provide a stronger interaction with the lysine - rich , positively charged binding pocket of hsa . on the other hand , the zwitterionic gd(5b ) we also measured the relaxivity for these complexes in a 4.5% hsa solution ( table 4 ) . at 20 mhz , the relaxivity of gd(4b ) increased over 5-fold when hsa was added , whereas for gd(5b ) , the increase was less than 3-fold . such a difference could not be accounted for by the difference in the fraction bound to albumin between the two complexes . in the absence of albumin , the relaxivity of gd(4b ) is about 25% higher than that of gd(5b ) , but in the presence of albumin , the relaxivity of gd(4b ) is 120% and 80% higher than that of gd(5b ) at 20 and 60 mhz , respectively ( table 4 ) . in order to better understand this phenomenon , we performed additional r1 measurements at different temperatures and additional field strengths . we acquired relaxivity data for gd(4b ) and gd(5b ) in pbs and 4.5% ( w / v ) hsa at 20 , 30 , 60 , 100 , 200 , and 400 mhz and at 10 , 25 , 37 , and 50 c ( tables s1s4 in the supporting information , si ) . we also measured the fraction ( fb ) of gd(4b ) and gd(5b ) bound to hsa at each of these temperatures ( table s5 in the si ) . because we know the fraction bound to hsa for each complex and we measure the relaxivity of the unbound complex independently , we can calculate the relaxivity due to the hsa - bound species ( r1bound ) for both gd(4b ) and gd(5b ) ( eq 1).1 the magnitude and field dependences of these hsa - bound relaxivities are shown in figure 3 . the high relaxivity of gd(4b ) could not be well described by a pure second - sphere model , which assumes some adjustable number of long - lived water molecules in close proximity to the metal ion . instead , we reasoned that the difference in the hsa - bound relaxivities between gd(4b ) and gd(5b ) was due to the presence of an inner - sphere water ligand on some fraction of the gd(4b ) complexes ( eq 2 ) . we assumed that the difference in the hsa - bound relaxivity between gd(4b ) and gd(5b ) was the contribution from this inner - sphere water ( eq 3 ) . we then calculated this difference at each temperature and field and simultaneously fit the resultant data set using solomon bloembergen morgan theory ( figure 3 and eqs 28 ) . while the hsa - bound relaxivity of gd(4b ) was higher than expected , it was not as high as one would anticipate for a complex with one coordinated water , and so we left the hydration number q as a temperature - dependent adjustable parameter defined by an equilibrium constant . we found that this entire data set could be reasonably well described ( figure 3 ) by five adjustable parameters : q ( mole fraction of q = 1 species at 310 k ) , h ( enthalpy change for equilibrium between q = 0 and 1 species ) , m , h ( activation energy for water exchange ) , and [ square of the trace of zero - field - splitting ( zfs ) tensor ; eqs 38].2345678we found that the data were relatively insensitive to the rotational correlation time as long as this correlation time was large . this is reasonable because the very short water residency time should dominate the correlation time for relaxation . in addition , the data were insensitive to v , the correlation time for electronic relaxation , and so we fixed this to 20 ps , in line with other studies of similar complexes . the quality of the fit could likely be improved by the addition of more adjustable parameters , e.g. , lipari szabo - type treatment , but the conclusions are unlikely to change . relaxivity of hsa - bound gd(5b ) ( filled circles ) and gd(4b ) ( open circles ) at ph 7.4 , at ( a ) 10 c , ( b ) 25 c , ( c ) 37 c , and ( d ) 50 c . the difference between the solid and open circles is the inner - sphere contribution to the gd(4b ) relaxivity , and the solid line is the fit to the data described in the text . the analysis shows that there is a 15% fraction of gd(4b ) that exists as a q = 1 complex with a short residence time for the first sphere water molecule . for the equilibrium in eq 2 , the two reactions are the formation and dissociation of the q = 1 complex . in principle , there can also be water exchange on the q = 1 complex . because we only observe free water , we can not distinguish between these two reactions ( dissociation to a stable q = 0 complex versus water exchange at the q = 1 complex ) . therefore , we likely are measuring the formation and dissociation of water to the gadolinium complex ; i.e. , m , the residence time in the bound state , corresponds to 1/kd . the relatively high relaxivity of gd(4b ) in the presence of hsa , especially at high fields , prompted us to further explore the kinetic inertness of this complex with respect to gadolinium(iii ) dissociation . acetate to methylenephosphonate substitution in lanthanide(iii ) polyaminocarboxylates typically results in a more thermodynamically stable complex ( because of the increased basicity of the phosphonate ) , but at the same time , the complex can become more kinetically labile . we measured the half - life of gd(4b ) under different forcing conditions and compared it directly to the acetate analogue gd(6 ) measured under the same conditions . in the first experiment , we challenged each complex with 1 equiv of ms-325 ligand [ a dtpa derivative of higher thermodynamic stability than gd(dotala ) derivatives ] at ph 3 and 37 c and monitored transchelation by hplc . under these conditions , we found no measurable decomplexation of gd(4b ) , while gd(6 ) underwent complete transchelation to ms-325 with a half - life of 115 h. this result suggests that gd(4b ) is much more thermodynamically stable than ms-325 . in a second experiment , we challenged each complex with a 100-fold excess of citrate at ph 3 and 37 c . with a citrate challenge , we found the opposite behavior : transchelation proceeded to completion in both cases but over 1 order of magnitude more rapidly in the case of gd(4b ) ( t1/2 = 9.5 h ) than gd(6 ) ( t1/2 = 110 h ) ( figure 4 ) . dechelation of gd(4b ) ( filled circles ) and gd(6 ) ( open circles ) at ph 3 and 37 c in the presence of 10 mm citrate . this is in agreement with the previously reported kinetic data obtained for acyclic phosphonate complexes compared to their acetate analogues . we hypothesize that the phosphonate arm promotes the formation of ternary complexes with citrate molecules , which facilitates decomplexation in the case of gd(4b ) . although gd(4b ) was more labile than gd(6 ) , we note that acyclic complexes like [ gd(dtpa)(h2o ) ] or its derivatives , such as [ gd(bopta)(h2o ) ] ( table s5 in the si ) , are much more labile under these conditions , e.g. , reaching transchelation equilibrium with ms-325 ligand in under 10 min . because the high field relaxivity of gd(4b ) was 50% higher than that of the approved contrast agent ms-325 at 4.7 t , we decided to assess its relaxation properties in vivo . in addition , both gd(4b ) and ms-325 showed equivalent affinity to hsa , making this comparison more direct . a catheter was placed in the tail vein so the mouse could be injected while positioned in the scanner . we performed 3d t1-weighted imaging before and immediately after injection of each compound ( 0.1 mmol kg ) . immediately after injection , the blood vessel enhancement observed with gd(4b ) was 38 2% higher than that observed with ms-325 ( figure 5 ) consistent with the higher relaxivity of the former . preinjection , as well as 1 min postinjection , coronal images obtained with ms-325 ( second from left ) and gd(4b ) ( right ) . gd(4b ) shows visibly better contrast in the vena cava , which can be quantified as 38 2% better contrast ( vs muscle ) . the dose of agent , time point , and image windowing was the same for both studies . kidney ( ki ) and vena cava ( vc ) are denoted in the far right image . we have synthesized and evaluated the properties of five different derivatives of a new phosphonate analogue ( dotalap ) of the single amino acid chelate dotala . on the basis of literature examples , we anticipated and observed an increased relaxivity due to the established second - sphere effect of the phosphonate . however , for [ gd(dotala)(h2o ) ] and gd(dotala)-propionamide derivatives , water exchange is already very fast ( m = 9 and 17 ns , respectively ) . for the gd(dotalap ) complexes investigated here , we found that further acceleration of water exchange through crowding of the coordination sphere may instead lead to stabilization of the q = 0 , 8-coordinate state . the five complexes investigated here comprised two that existed primarily as 9-coordinate q = 1 complexes , while the other three appear to be primarily q = 0 , presumably 8-coordinate complexes . these conclusions are supported by o nmr and h relaxivity data on the gadolinium(iii ) complexes and by luminescence lifetime measurements on the europium(iii ) complexes . the energy difference between the 8- and 9-coordinate states is quite small , and peripheral changes to the macrocyclic ligand outside the inner coordination sphere can alter the coordination number in a nonpredictable manner . this delicate equilibrium between q = 0 and rapidly exchanging q = 1 underlines a challenge in the design of gadolinium(iii ) complexes with extremely fast water exchange rates . two of the investigated derivatives , gd(4b ) and gd(5b ) , were designed to bind hsa . the relaxivities of these compounds in the absence of hsa were similar and low , although r1 for gd(4b ) was about 25% higher than r1 for gd(5b ) . this small increase in the relaxivity could be due to the presence of a small amount of q = 1 species for gd(4b ) . however , in the presence of hsa , the relaxivity difference between the complexes is magnified , and this increased difference seen when the molecular tumbling rate is decreased is indicative of a small fraction of q = 1 species . variable - temperature nmrd allowed us to estimate that about 15% of gd(4b ) exists as a q = 1 species at 37 c , at least while it is bound to albumin . the presence of this q = 1 complex whose coordinated water has an extremely short residency time results in remarkably high relaxivities at high field for a complex bound to hsa . for instance , the relaxivity at 4.7 t was 50% higher than that for the albumin - targeted approved agent ms-325 . the nmrd results have profound consequences for the development of slow tumbling contrast agents for high - field applications . the variable - temperature data clearly indicate that the water residency time , m , is the dominant correlation time for modulating the relaxivity in this system . figure 6 shows data at two temperatures and two fields to illustrate this point . at 0.47 t ( 20 mhz ) , m at 2.4 ns is too short for optimal relaxivity , but as the temperature is lowered , m increases and the relaxivity goes up . on the other hand , at higher fields ( > 2 t ) , the optimal correlation time is in the 13 ns range . at 37 c , a m of 2.4 ns is ideal for relaxivity at 2.4 t ( 100 mhz ) . however , as the temperature is lowered and m increases , the correlation time becomes too long for optimizing high - field relaxivity . the very short m provides increased relaxivity at high fields and offers a strategy to increase the relaxivity of slow tumbling entities at high magnetic fields . we note that the data in figure 6 are for a complex that is only 25% ( at 10 c ) or 15% ( at 37 c ) for q = 1 . thus , the relaxivity gains for a single q = 1 species are 46-fold higher . inner - sphere relaxivity of gd(4b ) bound to hsa at two different temperatures ( 10 and 37 c ) and larmor frequencies . because the relaxivity of q = 0.15 gd(4b ) was still clearly enhanced compared to q = 1 ms-325 at higher fields , we considered its evaluation for high - field in vivo imaging . for this , we first tested the kinetic inertness of gd(4b ) under forcing conditions . we found the somewhat decreased kinetic inertness , when compared with the all - acetato analogue gd(6 ) , is a typical reflection of the results previously reported in conjunction of phosphonato compounds . this encouraged the in vivo evaluation and cross - comparison with the approved hsa binding compound ms-325 . the subsequent in vivo imaging experiment at 4.7 t was found to mirror the in vitro results well because we were able to obtain better angiographic contrast for gd(4b ) compared to ms-325 . an increased blood signal is clearly found in the increased relaxivity for this compound at intermediate magnetic fields . we consider this outcome a promising result that encourages us to further investigate m acceleration as a way to enhance the relaxivity at higher fields because an entirely q = 1 complex with such a fast exchange rate would have 5 times the relaxivity observed with gd(4b ) . however , the somewhat unpredictable nature of the compound behavior in terms of the inner - sphere hydration number is a clear challenge . in summary , we have successfully synthesized a monophosphonate analogue of the single amino acid chelator dotala , which we term dotalap . the gd(dotalap ) derivatives show enhanced second - sphere relaxivity compared to gd(dotala ) . in two derivatives , we found increased water exchange kinetics , and this combination of fast water exchange and increased second - sphere relaxivity results in improved relaxometric behavior . we also found , however , that functionalization of this single amino acid chelate of either the nitrogen or carbon terminus with different r groups can sometimes yield gadolinium complexes with no inner - sphere water ( q = 0 ) or a mixture of q = 0 and 1 complexes . this decrease in the hydration number appears as a side effect of accelerated water exchange and does not correlate with a specific r group or location of functionalization . for one derivative , gd(4b ) , where r was chosen such that the corresponding gadolinium complex had affinity to hsa , we found that the hydration number was 0.15 in the presence of hsa , i.e. , a 6:1 mixture of q = 0 to q = 1 . the corresponding mean water residency time obtained for the albumin - bound complex was shorter than the rotational correlation time , which , in this case , leads to m becoming the dominant correlation time for relaxation . because m is considerably shorter than r , higher inner - sphere relaxivity was achieved for this complex at intermediate and high magnetic fields than what can be obtained with the clinically approved hsa targeting agent ms-325 . we have demonstrated the effect of higher relaxivity also in vivo with higher blood vessel to muscle contrast for gd(4b ) when compared to ms-325 . future endeavors will include the identification of fast exchanging complexes that are exclusively q = 1 in order to maximize the relaxivity gains at high fields . h and c nmr spectra were recorded on a varian 11.7 t nmr system equipped with a 5-mm broad - band probe . hplc purification of intermediates was performed on a rainin , dynamax ( phenomenex c18 column : 250 mm 21.2 mm , 10 m ) using method a : 0.1% trifluoroacetic acid ( tfa ) in water with a gradient of 595% ( 0.1% tfa in mecn ) over 20 min with a 15 ml min flow rate . hplc purity analyses ( both uv and ms detection ) were carried out on an agilent 1100 system ( phenomenex luna c18(2 ) column : 100 mm 2 mm , 0.8 ml min flow rate ) with uv detection at 220 , 254 , and 280 nm and positive - mode electrospray ionization ( esi ) using the following methods . method b : solvent a = water , solvent b = mecn ; 260% b over 15 min . method c : solvent a = 10 mm nh4oac , solvent b = 10% 10 mm nh4oac , 90% mecn ; 595% b over 15 min . method d : solvent a = 10 mm nh4oac , solvent b = 10% 10 mm nh4oac , 90% mecn ; 535% b over 15 min . chemicals were supplied by aldrich chemical co. , inc . , and were used without further purification . solvents ( hplc grade ) were purchased from various commercial suppliers and used as received . the monoalkylated cyclen precursor to compound 1 tri - tert - butyl phosphite was synthesized according to a procedure reported by manning et al . amide coupling followed by deprotection of the tert - butyl protective groups was done by activation of the carboxylate with hatu ( 1.2 equiv ) and dipea ( 1.2 equiv ) for 5 min in n , n - dimethylformamide ( dmf ) , followed by the addition of amine ( 1 equiv ) dissolved in dmf and stirring at room temperature for 18 h. after confirmation of the presence of the amide - coupling product by lc ms ( method b ) , the intermediate was isolated by preparative hplc ( method a ) , eluting between 11 and 13 min . the clean fractions containing the desired product were pooled and lyophilized to afford the intermediate as an off - white powder in 1535% yield . redissolution of intermediates described above in a 1:1 mixture of dichloromethane and tfa , followed by stirring for 18 h , afforded the final ligand with quantitative yield . an amount of a stock solution containing lncl36h2o ( 0.95 equiv based on the ligand weight ) is added to the ligand solution under monitoring of the ph . the lightly cloudy solution is filtered and lyophilized to afford the corresponding lanthanide complex as an off - white powder . ms ; the presence of free lanthanides was excluded using the xylenol orange test . 2-[[(benzyloxy)carbonyl]amino]-3-(1,4,7,10-tetraazacyclododecan-1-yl)propanoate ( 1.475 g , 3.05 mmol , 1 equiv ) and paraformaldehyde ( 0.34 g , 11.45 mmol , 3.75 equiv ) were dissolved in tetrahydrofuran and vigorously stirred under a dinitrogen atmosphere for 0.5 h. tri - tert - butyl phosphite ( 1.145 g , 4.5 mmol , 1.5 equiv ) was then added , and the reaction was stirred for 16 h. reaction monitoring by lc ms showed the product as [ m + h ] ( m / z 690.4 ) . the reaction mixture was then filtered , concentrated in vacuo , and redissolved in etoac ( 200 ml ) . the organic layer is washed with 50 ml of saturated na2co3 and 50 ml of brine , dried with sodium sulfate , and concentrated to afford the dialkylated intermediate ( 1.73 g , 2.5 mmol , 82% ) , which was used in the subsequent step without further characterization . k2co3 ( 0.68 g , 4.9 mmol , 2 equiv ) was added , and the mixture was stirred vigorously after the addition of tert - butyl bromoacetate ( 0.766 g , 0.575 ml , 3.94 mmol , 1.6 equiv ) . after 16 h , water ( 70 ml ) was added and most of the mecn was removed in vacuo . the oily residue was taken up in etoac ( 80 ml ) and washed with water and brine ( 100 ml each ) . the organic layer was separated , dried with sodium sulfate , and concentrated to afford the crude product , which was redissolved in mecn ( 5 ml ) and purified using preparative hplc ( method a ) . fractions containing the product are pooled and lyophilized to afford the product as a white powder ( 0.43 g , 0.46 mmol , 20% yield ) . h nmr ( cdcl3 , 500 mhz , ppm ) : 9.9 ( s , br , 2h ) , 7.367.34 ( m , 10h ) , 5.255.16 ( m , 4h ) , 4.3 ( m , 1h ) , 3.352.68 ( m , 21h ) , 2.04 ( s , 4h ) , 1.511.43 ( m , 36h ) . c nmr ( cdcl3 , 125.7 mhz , ppm ) : 175.4 , 170.6 , 170.3 , 135.0 , 131.1 , 129.4 , 128.6127.9 , 81.4 , 81.0 , 67.7 , 57.347.4 , 30.6 , 28.2 , 22.1 . calcd for c47h76n5o11p4 : m / z 917.5 . found : m / z 918.6 ( [ m + h ] ) . compound 1 ( 0.43 g , 0.47 mmol ) was dissolved in etoh ( 40 ml ) . pd / c [ 10% ( w / v ) , 0.215 g ] was added , and the reaction mixture was purged first with dinitrogen and then charged with dihydrogen ( 1 atm ) . the mixture was then stirred under a dihydrogen atmosphere for 3 h , after which the reaction was found to be complete . the pd / c was removed by filtration , and the solvent was removed in vacuo to afford the product as a colorless oil ( 0.32 g , 0.46 mmol , quantitative conversion ) . h nmr ( cd3od , 500 mhz , ppm ) : 5.15 ( s , br , 1h ) , 4.462.48 ( m , 24h ) , 1.631.47 ( m , 36h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 167.9 , 160.5 , 86.6 , 82.6 , 55.248.5 , 30.6 , 29.2 , 26.9 . calcd for c32h64n5o9p : m / z 693.4 . found : m / z 694.7 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 5.15 ( s , br , 2h ) , 4.262.46 ( m , 25h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 175.1 , 169.6 , 55.448.9 . calcd for c16h32n5o9p : m / z 469.2 found : m / z 470.1 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 7.387.23 ( m , 5h ) , 5.15 ( s , br , 1h ) , 4.76 ( m , 1h ) , 3.982.86 ( m , 24h ) , 1.47 ( t , 3h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 159.4 , 140.9 , 128.5126.8 , 116.7 , 114.4 , 53.3 , 49.5 , 46.1 , 17.1 . calcd for c25h40n5o10p : m / z 601.2 . found : m / z 602.4 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 7.28 ( d , 2h ) , 7.20 ( d , 2h ) , 4.73 ( s , br , 1h ) , 3.912.87 ( m , 24h ) , 2.45 ( m , 2h ) , 1.84 ( m , 1h ) , 1.481.29 ( m , 5h ) , 0.89 ( d , 6h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 172.8 , 169.6 , 140.5 , 138.2 , 129.5 , 127.0 , 54.748.2 , 30.0 , 21.3 , 17.1 . calcd for c28h48n5o9p : m / z 629.3 . found : m / z 630.5 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 7.397.26 ( m , 5h ) , 5.02 ( s , br , 1h ) , 4.152.46 ( m , 24h ) , 1.641.46 ( m , 3h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 169.7 , 166.3 , 160.4 , 142.7 , 128.5125.8 , 119.6 , 117.3 , 114.9 , 112.7 , 53.349.5 , 27.2 , 20.85 . calcd for c24h41n6o8p : m / z 573.3 . found : m / z 574.6 ( [ m + h ] ) . h nmr ( cd3od , 500 mhz , ppm ) : 7.24 ( d , 2h ) , 7.10 ( d , 2h ) , 4.212.44 ( m , 30h ) , 1.941.29 ( m , 6h ) , 0.89 ( d , 6h ) . c nmr ( cd3od , 125.7 mhz , ppm ) : 175.4 , 169.8 , 167.4 , 140.2 , 138.8 , 128.9 , 126.7 , 54.648.1 , 38.2 , 30.1 , 21.3 , 17.1 . p nmr ( cd3od , 202.4 mhz , ppm ) : 6.1 . lc calcd for c31h54n7o9p : m / z 699.3 . found : m / z 700.6 ( [ m + h ] ) . calcd for c16h28gdn5o9p [ gd(3 ) ] : m / z 623.1 . found : m / z 625.1 ( [ m + 2h ] ) . . found : m / z 620.1 ( [ m + 2h ] ) . . found : m / z 757.2 ( [ m + 2h ] ) . calcd for c24h36eun5o9p [ eu(4a ) ] : m / z 750.1 . found : m / z 752.1 ( [ m + 2h ] ) . calcd for c29h44gdn5o10p [ gd(4b ) ] : m / z 811.2 . found : m / z 813.4 ( [ m + 2h ] ) . calcd for c29h44eun5o10p [ eu(4b ) ] : m / z 806.2 . found : m / z 808.4 ( [ m + 2h ] ) . calcd for c29h44tbn5o10p [ tb(4b ) ] : 812.2 . found : m / z 814.4 ( [ m + 2h ] ) . calcd for c24h38gdn6o8p [ gd(5a ) ] : m / z 727.2 . found : m / z 728.2 ( [ m + h ] ) . calcd for c24h38eun6o8p [ eu(5a ) ] : m / z 722.2 . found : m / z 723.3 ( [ m + h ] ) . calcd for c31h51gdn7o9p [ gd(5b ) ] : m / z 852.3 . found : m / z 853.5 ( [ m + h ] ) . calcd for c31h51eun7o9p [ eu(5b ) ] : m / z 849.3 . found : m / z 850.4 ( [ m + h ] ) . luminescence lifetime measurements of europium complexes in h2o and d2o were performed on a hitachi f-4500 fluorescence spectrophotometer . for the measurements in d2o , the complexes were first dissolved in d2o ( 99.98% d ) , lyophilized , and dissolved in d2o again to reduce the amount of residual hdo . measurements were taken with the following settings : excitation at 396 nm ( eu ) emission at 616 nm ( eu ) 30 replicates , 0.04 ms temporal resolution ( 020 ms ) , pmt voltage = 400 v. lifetimes were obtained from monoexponential fits of the data using igor pro software ( version 6.0 , wavemetrics , lake oswego , or ) . o nmr measurements of solutions were performed at 11.7 t on 350 l samples contained in 5 mm standard nmr tubes on a varian spectrometer . the temperature was regulated by an air flow controlled by a varian vt unit . the h2o transverse relaxation times of a > 4 mm solution of all gadolinium complexes ( ph 7.4 , 10 mm pbs buffer ) were measured using a cpmg sequence . the exact concentration of the sample was determined by inductively coupled plasma mass spectrometry ( icp - ms ) . reduced relaxation rates , 1/t2r , were calculated from the difference of 1/t2 between the sample and water blank and then divided by the mole fraction of coordinated water , assuming q = 1 . the temperature dependence of 1/t2r was fit to a four - parameter model as previously described . the gd o hyperfine coupling constant , a/ , was assumed to be 3.8 10 rad s. ultrafiltration measurements of protein binding solutions of a 0.1 mm gadolinium compound and 4.5% hsa ( 200 l total ) were added to a 5000 mw ultrafiltration unit ( regenerated cellulose membrane , millipore , bedford , ma ) . the samples were incubated at a set temperature for 30 min and subsequently centrifuged to filter off the nonprotein - bound components . the concentration of bound complex in the protein samples was determined by subtracting the gadolinium concentration ( unbound complex ) found in the filtrate from the gadolinium concentration of the initial solution . stock solutions of ms-325-l ( the ligand of the ms-325 complex ) , gd(4b ) , and gd(6 ) were prepared . for experiment 1 , ms-325-l was added to solutions of the gadolinium complexes in ph 3 water and incubated at 37 c . a 10 l aliquot was removed for hplc analysis and analyzed at time points 1 , 20 , 50 , 72 , 80 , 96 , 120,168 , 192 , 216 , 240 , 288 , 312 , 336 , 360 , and 480 h , while the remainder of the solution was incubated at 37 c . for experiment 2 , the gadolinium complexes were added to a 10 mm ph 3 citrate buffer , affording a 0.1 mm complex concentration , and incubated at 37 c . a 10 l aliquot was removed and analyzed at 0 , 0.75 , 1.5 , 2 , 3 , 4 , 5 , 6 , 24 , 72 , 96 , 120 , 168 , 192 , 216 , 240 , 288 312 , 336 , and 408 h. nu / nu mice were anesthetized with isoflurane ( 12% ) and placed in a specially designed cradle with the body temperature maintained at 37 c . the tail vein was cannulated for intravenous delivery of the contrast agent while the animal was positioned in the scanner . imaging was performed at 4.7 t , using a custom - built volume coil to acquire coronal t1-weighted images with a field of view that covered the major organs of the thorax and abdomen ( heart , stomach , liver , intestines , and kidney ) . t1-weighted images were acquired using a 3d fast low angle shot ( flash ) sequence : tr / te / flip angle = 15.3 ms/1.54 ms/40 , fov = 4.8 2.4 2.4 cm , matrix = 192 96 96 , 1 average . in the previous issue of critical care , kmpers and colleagues demonstrated a direct correlation between increased peripheral blood levels of the vascular growth factor , angiopoietin ( angpt)-2 , and mortality in 43 critically ill adults with sepsis . endothelial injury is one of the main hallmarks of sepsis , leading to capillary leak , microcirculatory dysfunction , organ failure , and eventual death in many critically ill patients . angpt-1 and angpt-2 are two of the best - characterized members of a family of endothelial - derived vascular growth factors necessary for both normal and pathologic angiogenesis and vasculogenesis . both angpt-1 and angpt-2 appear to bind to the tyrosine kinase receptor , tie-2 , found primarily on the luminal surface of endothelial cells . recent studies have also shown that the tie-2 receptor may be found on certain populations of peripheral blood monocytes , although the function and role of the tie-2 receptor in the host innate immune response remain relatively unexplored . angpt-1 is a tie-2 agonist and promotes endothelial stabilization and quiescence , whereas angpt-2 is a tie-2 antagonist and promotes endothelial activation , destabilization , and inflammation . as such , the relative balance between angpt-2 and angpt-1 at the tie-2 receptor may be more relevant to the pathobiology of sepsis than the absolute levels of the individual growth factors . several studies have demonstrated increased peripheral blood levels of angpt-2 in critically ill patients with sepsis [ 5,7 - 9 ] , multiple trauma , acute lung injury ( ali ) , and cardiopulmonary bypass when compared with healthy controls . more importantly , increased angpt-2 levels appear to be associated with adverse outcomes [ 5,6,9 - 12 ] . for example , the study of kmpers and colleagues showed that increased peripheral blood angpt-2 levels correlated with surrogate markers of tissue hypoxia , disease severity , and mortality in 43 critically ill adults with sepsis . also of note , consistent with the opposing roles of angpt-2 and angpt-1 on the tie-2 receptor , peripheral blood levels of angpt-1 were significantly lower in the patients with sepsis compared with healthy controls . unfortunately , in the study of kmpers and colleagues , similar to the aforementioned studies , the temporal kinetics of angpt-1 and angpt-2 were not assessed as blood samples were collected upon the first day of admission to the intensive care unit only . angpt-2 is stored in the weibel - palade bodies within endothelial cells in a more or less prepackaged form . it is therefore not surprising that angpt-2 levels are increased early in response to endothelial activation or injury . whether angpt-2 levels remain increased in critically ill patients with sepsis has not been directly addressed and is a question for future investigation . it is certainly tempting to speculate that peripheral blood angpt-2 levels would be an ideal biomarker of early endothelial activation and injury . similarly , whether angpt-1 levels remain decreased in critically ill patients who eventually succumb to their illness is an interesting question . angpt-1 may be a biomarker of endothelial recovery ; however , given its purported anti - inflammatory role , angpt-1 would appear to be an attractive therapeutic target as well . to this end , several studies have suggested that manipulating the ratio of angpt-2 to angpt-1 by augmenting angpt-1 levels may represent an ideal therapeutic strategy for patients with sepsis and ali . important translational laboratory studies are necessary to show that increased angpt-2 levels in critically ill patients are more than just an epiphenomenon . the role of angpt-2 in the pathobiology of sepsis and ali needs to be further elucidated by using in vitro cell - based studies and animal models of critical illness . similarly , the presence of the tie-2 receptor on certain subpopulations of peripheral blood monocytes suggests a larger role for angpt-2 in the host innate immune response . finally , manipulation of the angpt / tie-2 system may be a rational therapeutic strategy for the management of critically ill patients with sepsis and ali . all of these questions remain an active focus in several laboratories , including our own . the authors ' research is funded by the national institutes of health ( bethesda , md , usa ) ( grant numbers 5ko8gm077432 and 1r03hd058246 ) .

here , we describe the synthesis of the single amino acid chelator dotalap and four of its derivatives . the corresponding gadolinium(iii ) complexes were investigated for their kinetic inertness , relaxometric properties at a range of fields and temperatures , water exchange rate , and interaction with human serum albumin ( hsa ) . derivatives with one inner - sphere water ( q = 1 ) were determined to have a mean water residency time between 8 and 6 ns in phoshate - buffered saline at 37 c . the corresponding europium complexes were also formed and used to obtain information on the hydration number of the corresponding coordination complexes . two complexes capable of binding hsa were also synthesized , of which one , gd(5b ) , contains no inner - sphere water , while the other derivative , gd(4b ) , is a mixture of ca . 15% q = 1 and 85% q = 0 . in the presence of hsa , the latter displayed a very short mean water residency time ( m310 = 2.4 ns ) and enhanced relaxivity at intermediate and high fields . the kinetic inertness of gd(4b ) with respect to complex dissociation was decreased compared to its dotala analogue but still 100-fold more inert than [ gd(bopta)(h2o)]2. magnetic resonance imaging in mice showed that gd(4b ) was able to provide 38% better vessel to muscle contrast compared to the clinically used hsa binding agent ms-325 . the early recognition and management of sepsis remain the greatest challenges in the field of critical care medicine . endothelial injury is one of the hallmarks of sepsis , leading to capillary leak , microcirculatory dysfunction , organ failure , and eventual death in many critically ill patients . the angiogenic growth factors , angiopoietin ( angpt)-1 and angpt-2 , act upon the tie-2 receptor in opposing roles . angpt-2 has been found in abundance in septic patients when compared with healthy controls . in the study by kmpers and colleagues in the previous issue of critical care , angpt-2 levels correlated with markers of tissue hypoxia , disease severity , and mortality in septic adults . however , the temporal kinetics of the angiopoietins were not assessed . it remains to be seen whether angpt-2 levels will function solely as an early marker of sepsis or whether the manipulation of the angpt / tie-2 system will become a rational therapeutic target for the management of sepsis .

we compared monthly listeriosis data from england and wales with temperature records from 1989 through 2007 to determine the influence of various potential predictors on the number of listeriosis cases . uk health protection agency ( hpa ) data listing total monthly cases of human listeriosis in england and wales during 19902007 ( 4,5 ) are aggregate . all age categories and regions were included and were collated by the hpa centre for infections from voluntary reporting by microbiology laboratories and from referrals of cultures . these publicly available data were also validated by the hpa , and in our analysis we used revised figures based on that validation . our analysis covered the period from 1990 , when active surveillance of listeriosis began , through 2007 . undated cases that could not be assigned to a particular month were excluded from analysis . we used the uk met office mean monthly area temperature time series for 19892007 and 30-year means averaged during 19611990 ( 6 ) . exploratory linear regression analyses suggested a positive correlation between the number of listeriosis cases and the monthly mean uk ambient temperature , as well as suggesting a change in this relationship after 2000 ( p = 0.001 ; figure 1 , panel a ) . however , residual variability was not constant , and the monthly counts are likely to be overdispersed due to clustering of cases ( 3 ) . the data were fitted again by using a negative binomial generalized linear model with a logarithmic link function , a common model for time series of foodborne illness cases ( 7,8 ) . to separate seasonality of listeriosis rates from dependence on temperature , we considered the 30-year mean monthly temperatures from 19611990 , as well as monthly temperature anomalies ( observed mean temperature minus 30-year mean ) . to determine whether temperatures could have a delayed effect on listeriosis incidence , we also included mean and anomaly temperature variables lagged by 1 or more months . to allow for 2 break points at which the incidence of listeriosis may have suddenly changed , dummy variables were used to represent periods before , between , and after the months in which these increases might have occurred . a best - fit model was selected according to the corrected akaike information criterion ( aic ) ( 9 ) by using stepwise regression at all combinations of 2 break point months from january 1996 through december 2007 . to examine changing effects on incidence , interactions between break point indicators and other variables were considered , even if the main effects were not yet in the model . main effects were subsequently added to the final model if any interaction terms were included . a ) listeriosis cases compared with mean observed monthly uk temperatures , 19902000 ( triangles ) and 20012007 ( circles ) . shown are an increased overall incidence in 20012007 ( dashed line ) versus 19902000 ( solid line ) and a significant change in the linear dependence of incidence on temperature ( p = 0.001 ) . b ) best - fit pair of break points and other pairs of break points with support , according to the corrected akaike information criterion ( aic ) . the 2 break - point months are varied to find the lowest value ( black square ) . pairs of break points with good support relative to the best model ( corrected aic within 4 of the best fit ; dark gray squares ) or moderate to weak support ( corrected aic within 10 of the best fit ; light gray squares ) are also shown . pairs of break points with little or no support ( corrected aic > 10 greater than the best fit ; white area ) include those models for which only 1 break point exists ( squares along the dashed line ) . the best - fit model included 2 break points ( tables 1 , 2 ; figure 1 , panel b ; figure 2 ) . the overall rate of cases increased by 40.83% in april 2001 ( adjusted p = 0.001 , 95% confidence interval [ ci ] 17.29%68.93% ) ; a further increase of 47.76% occurred in march 2003 ( adjusted p<0.001 , 95% ci 27.92%71.13% ) . according to the corrected aic , there is statistical evidence of changes in the incidence of listeriosis in a range of months around these best - fit values , and weak support for a change in early 2005 , apparently due to low numbers of cases in the first months of that year and the following winter ( figure 1 , panel b ) . most of the seasonality in the number of cases was accounted for by the 30-year mean monthly temperatures . each extra degree celsius of mean monthly temperature corresponded to a 2.42% increase in cases in the current month ( adjusted p = 0.044 , 95% ci , 0.55%4.33% ) and a 4.09% increase in the following month ( adjusted p<0.001 , 95% ci 2.19%6.03% ) . the 1-month lag reflects delays between food production and consumption plus the known long incubation of listeria infections . a relationship also appears to exist between the rate of listeriosis and the 1-month lagged temperature anomaly starting in april 2001 , which corresponded to an additional 5.71% of listeriosis cases per degree celsius ( 95% ci 2.10%14.15% ) when compared with the main effect of temperature anomaly in 19902007 . this increase was not significant ( adjusted p = 0.312 ) , although the power of this test is only 15% at an adjusted significance level of 0.05 ( post hoc power analysis performed by computer simulation of the fitted model in the r statistical computing environment ) . the model also includes a slight negative trend in cases over time ( 0.13% per month , 95% ci 0.26%0.01% ) , but evidence for this trend is very weak ( adjusted p = 0.188 ) . * p values are for 2-sided tests of significance of the difference of each coefficient from zero ; adjusted p values are according to holm s method ( 10 ) . percentage changes in the main text can be derived from these coefficients by taking exponentials , subtracting 1 , and multiplying by 100 . the monthly number of listeriosis cases ( circles , lower plot ) is strongly seasonal , with a close relationship to the monthly mean temperature ( solid line , upper plot ) . overall listeriosis incidence per month underwent 2 sudden increases , at break points around april 2001 and march 2003 . our fitted statistical model ( dashed line , lower plot ) provides a close fit to the observed data ; the seasonally - adjusted fitted model ( solid line , lower plot ) shows the large magnitudes of the jumps in the rate of cases at the 2 break points . the changes in the incidence of listeriosis appear to have been quite sudden ( figure 2 ) . causative explanations based on gradual demographic or behavioral changes have previously been ruled out ( 3 ) , and those based on dynamic processes seem unlikely because no evidence exists for epidemiologic feedback between the source of infections and clinical cases . one possibility is that the increases are due to contamination of a small number of food products , as has been suggested as an explanation for an upsurge in listeriosis rates in the late 1980s . however , a restricted range of strains was responsible for most of the additional cases at that time ( 12 ) , and no evidence exists of such a pattern since 2001 ( 3 ) . an alternative possibility is that the phenomenon is a consequence of changes in government policy or business practices that have had more widespread effects on food processing , distribution , or preparation . we found notable coincidences between the dates of the increases in listeriosis infection rates and the dates of events associated with the 2001 foot and mouth disease ( fmd ) outbreak in the united kingdom . the april 2001 increase in listeriosis rates occurred shortly after the outbreak of fmd in february 2001 , allowing for a delay similar to the incubation period of listeria infections . the march 2003 increase in listeriosis rates occurred in the same month as the relaxation of movement restrictions on livestock instituted after the 2001 fmd outbreak ( 13 ) the diverse and widespread consequences of the 2001 fmd outbreak are well - documented ( 14,15 ) , and it seems plausible that such major disturbances to agricultural production could be the ultimate cause of the large increases in listeriosis rates that have been observed . the coincidence of these events raises the possibility that the change in listeriosis rates would be an unrecognized outcome of the 2001 uk fmd crisis , although we caution that our analysis of the of these cases does not demonstrate whether a causative link exists . listeriosis incidence in england and wales has increased notably since the beginning of 2001 , with 2 separate , sudden increases recorded in april 2001 and march 2003 . gillespie et al . argue that blame for these increases can not be ascribed to any of a variety of specific factors ( 3 ) . instead , more widespread changes affecting food production , processing , or consumption could be the root of the problem . incidence of this serious disease has risen substantially in england and wales , and an understanding of why will be important for management of listeriosis as a public health issue . attachment is a relatively stable emotional bond which is created between child and mother or those with whom an infant regularly interacts . parents responses to the signs of child 's attachment behavior and their availability in stressful situations , provides a safe place and condition for children , based on which , children organize their expectations from the environment . the attachment between child and primary caregiver ( usually mother ) would become internalized and later act as a mental model which is used by the adult person to use as a base for building friendship and romantic relationships ; it can affect the attitudes of people in their adulthood as well . adult attachment styles are subdivided into three categories : ( 1 ) secure : secure people are intimate and comfortable in making relationships , and they are sure that others would like them . ( 2 ) anxious - ambivalent : they have a strong desire for close relationships but also have many concerns of rejection . these people have a negative image of themselves , but a positive attitude toward others . ( 3 ) avoidance : for this group of people , self - reliance is the most valuable issue . hence , it can be said that attachment styles affect other aspects of one 's life and have an impact on persons relationships with other people after childhood . many researchers and authorities have shifted their focus toward the topics such as joy , happiness , life satisfaction , and positive emotions . according to many theories of emotions , one of the six great emotions is happiness ; the six great emotions include surprise , fear , anger , happiness , disgust , and worry . happiness is a type of conception about individual 's own life ; it includes items such as life satisfaction , positive emotions , and mood , lack of anxiety and depression and its different aspects of emotions . when people are satisfied with their living conditions and are frequently experiencing positive and less negative emotions , it is said that they are at high levels of mental health . increased levels of happiness is directly associated with the better status of health , appetite , sleep , memory , family relationships , friendships , family status , and ultimately mental health . the relationship between subjective well - being and emotion regulation with attachment styles in various studies has been explained . despite the important role of medical students in public health and the significance of their happiness which is related to their attachment styles , so far , this research was aimed to assess the relationship between attachment styles and happiness and demographic characteristics of medical students . this descriptive and analytical study was conducted on medical students in kurdistan university of medical sciences , in 2012 . as exclusion criteria , students who were unwilling to fill out a questionnaire and guest students since there were five independent variables in the study and it was needed to include 35 samples for each variable in the regression model , the calculated sample size was 175 people ; a total of 200 students were included in the study . samples were chosen through stratified sampling method ( different levels of education ) and each stratum was proportional to the size of each class . to collect the data , after obtaining permission from the ethics committee of kurdistan university of medical sciences , list of all medical students , which was classified by educational level , was obtained from education office . the samples were systematically selected from the list provided by education office ; they were selected in proportion to the number of students in each educational level ( physiopathology , extern , intern level ) . after taking their consent to participate in research and explaining the objectives , questionnaires were given to the participants . the questionnaires were filled out by the students and were collected the same day . before completing the questionnaire ( 47 questions ) , students were assured that all information will be confidential , and they were also asked to answer the questions accurately . they were allowed to ask their questions in case of facing any ambiguity in the questionnaire . this scale is developed by hazan and shaver ( 1987 ) and it has 15 items , with five items for each of the three types of secure attachment , ambivalent attachment , and avoidant attachment style . it is scored from never ( zero ) to almost always ( score = 4 ) . the score of each attachment subscale is obtained by calculating the mean of five items for each subscale . in various studies , the reliability of the questionnaire has been calculated from 0.78 to 0.81 ; moreover , its reliability in iranian culture was tested by boogar et al . , the obtained results for the entire test , the ambivalent , avoidant , and secure attachment styles were 0.75 , 0.83 , 0.81 , and 0.77 , respectively . to measure the happiness variable , the scale has 29 items which is scored on a range of zero to four ; it has five marks including life satisfaction with eight items , self - esteem with seven items , subjective well - being with five items , satisfaction with four items , and positive manner with three items . because two items have a correlation coefficients of < 35% with any of the five other components , they are not included in any of the components , but they are included in the total score . the reliability of this scale among iranian students has been reported to be 0.93 . the collected data were entered into spss version 16 ( ibm , chicago il , usa ) . quantitative data were described using the mean and standard deviation ( sd ) , and string variables were described using frequency and percentage . the correlation between happiness score and attachment style scores were assessed using pearson 's correlation coefficient . the difference between the happiness score and the scores of different attachment styles in each sex were compared using independent tests . the scores for different educational levels were compared using one - way anova . finally , using multiple regressions ( enter method ) , happiness variable as the dependent variable and the score of different attachment styles , gender , educational level , and grade point average ( gpa ) as the independent variables , if applicable , were entered into the model . this scale is developed by hazan and shaver ( 1987 ) and it has 15 items , with five items for each of the three types of secure attachment , ambivalent attachment , and avoidant attachment style . it is scored from never ( zero ) to almost always ( score = 4 ) . the score of each attachment subscale is obtained by calculating the mean of five items for each subscale . in various studies , the reliability of the questionnaire has been calculated from 0.78 to 0.81 ; moreover , its reliability in iranian culture was tested by boogar et al . , the obtained results for the entire test , the ambivalent , avoidant , and secure attachment styles were 0.75 , 0.83 , 0.81 , and 0.77 , respectively . to measure the happiness variable , the scale has 29 items which is scored on a range of zero to four ; it has five marks including life satisfaction with eight items , self - esteem with seven items , subjective well - being with five items , satisfaction with four items , and positive manner with three items . because two items have a correlation coefficients of < 35% with any of the five other components , they are not included in any of the components , but they are included in the total score . this scale is developed by hazan and shaver ( 1987 ) and it has 15 items , with five items for each of the three types of secure attachment , ambivalent attachment , and avoidant attachment style . it is scored from never ( zero ) to almost always ( score = 4 ) . the score of each attachment subscale is obtained by calculating the mean of five items for each subscale . in various studies , the reliability of the questionnaire has been calculated from 0.78 to 0.81 ; moreover , its reliability in iranian culture was tested by boogar et al . , the obtained results for the entire test , the ambivalent , avoidant , and secure attachment styles were 0.75 , 0.83 , 0.81 , and 0.77 , respectively . to measure the happiness variable , the revised oxford happiness inventory was used which had an overall reliability of 0.91 . the scale has 29 items which is scored on a range of zero to four ; it has five marks including life satisfaction with eight items , self - esteem with seven items , subjective well - being with five items , satisfaction with four items , and positive manner with three items . because two items have a correlation coefficients of < 35% with any of the five other components , they are not included in any of the components , but they are included in the total score . the collected data were entered into spss version 16 ( ibm , chicago il , usa ) . quantitative data were described using the mean and standard deviation ( sd ) , and string variables were described using frequency and percentage . the correlation between happiness score and attachment style scores were assessed using pearson 's correlation coefficient . the difference between the happiness score and the scores of different attachment styles in each sex were compared using independent tests . the scores for different educational levels were compared using one - way anova . finally , using multiple regressions ( enter method ) , happiness variable as the dependent variable and the score of different attachment styles , gender , educational level , and grade point average ( gpa ) as the independent variables , if applicable , were entered into the model . the mean ( sd ) of participants age was 22.42 ( 2.45 ) years . of all , 122 students ( 61% ) were female and 185 persons ( 92.5% ) were single . a total of 89 students ( 44.5% ) were in basic sciences educational level and the majority of participants , i.e. , 97 students ( 48.5% ) had gpa of 1517 [ table 1 ] . the distribution of demographic variables in studied subjects overall , the mean ( sd ) score of happiness was 62.71 ( 17.61 ) , secure attachment style was 11.46 ( 2.56 ) , avoidant attachment style was 9.34 ( 3.32 ) , and ambivalent attachment style was 7.93 ( 3.47 ) . there was no significant relationship between gender and attachment styles , however , the happiness score was 67.2 ( 17.2 ) in men and 59.9 ( 17.36 ) in women , and the difference was statistically significant ( p = 0.005 ) . the avoidant attachment style was 9.48 ( 3.34 ) in singles and 7.6 ( 2.66 ) in married people , and the difference was also statistically significant ( p = 0.03 ) [ table 2 ] . the relationship between gender and marital status of the studied subjects with attachment styles and happiness scores there was no significant relationship between the happiness score and educational level . the score of secure attachment style in students with gpa of 1720 was about 9.91 ( 2.9 ) , which was lower compared to those with lower gpas ( p = 0.051 ) . no significant relationship was observed between happiness score and other attachment styles with students gpas . age was not significantly correlated with happiness scores ( p = 0.797 , r = 0.019 ) . in the multivariate analysis , the relationship between attachment styles and happiness scores were compared and the results showed that after controlling for important factors , the variables of secure attachment style ( p = 0.001 ) , male gender ( p = 0.004 ) , and gpa ( p = 0.047 ) were associated with higher happiness scores ( r = 0.180 ) [ table 3 ] . comparison of the relationship between happiness scores and attachment style and other variables using multiple regression analysis the most common attachment style among students was secure attachment style that was consistent with the results of other studies . secure attachment style leads to activation of a system which bowlby calls the discovery system . this system allows a person to explore his / her environment and experience its own ability to control the condition . secure attachment gradually creates a sense of mastery and ability to handle frustration , and finally , in the context of a secure attachment relationship , then the person is enabled to reflect his / her emotions and positive beliefs about personal values and effectiveness . positive perfectionism , self - esteem , personal control , greater happiness in relationships better emotional management , less stress , and greater job satisfaction are among the specifications of secure attachment style ; these features may be a positive prognostic factor in medical students who usually endure much stress . in our study , the minimum frequency was observed in ambivalent attachment style ; our finding was similar to other studies . in asgharinejad et al.s study as well as ahadi et al.s study avoidant attachment style was the most common and secure attachment was second common style . due to differences in statistical samples and scales , which have been used in these two studies , these differences can be justified . attachment theory is focused on cognitive schema ; the schema affects the organization of individual 's relations with others and his / her perceptions of the world around . attachments formed in childhood can affect adulthood and the attachment between child and primary caregiver ( usually mother ) is internalized and serves as a mental model . according to the mentioned explanations , we can conclude that attachment styles are formed based on schemas and inner experiences , experiences which obtained through interaction with parents and others over time , the role of these factors is much stronger than the effect of gender alone . according to our results , there was a significant relationship between avoidant attachment style and marital status , and avoidant attachment style was more common among single people than married ; so , avoidant attachment could be a barrier to marriage . finney and noler believe that adults with avoidant attachment style have the same characteristics as those with dismissive attachment style ( self - positive model , others negative , with a low anxiety , and high avoidance ) . people with avoidant attachment styles have a negative attitude toward others and have difficulty in communicating with others and maintaining relationships ; they have a high sense of self - esteem and put low values on close relationships with others , which confirms our findings . the results showed no significant relationship between attachment style and gpa of individuals ; however , secure attachment style was less common in participants with high gpa . individuals with a secure attachment style are better able to interact with the environment , so they are expected to have better educational status , but the results of our study did not confirm this idea . it might be that struggling to get a higher score , sometimes help individual to compensate for a sense of frustration and low self - control . it is also possible that the educational system would create an unhealthy competitive environment and promote negative behaviors such as blind imitation without critical thinking . on the other hand , in our study , it was not determined to which educational level and age range each gpa belongs . in addition , the effects of other factors were not considered , and they have not even been considered in other studies as well , and this is one of the limitations of our study . in sheikhmoonesi et al.s study the average score of subjects in the happiness inventory was 41.23 and the average score of happiness in students of tehran university of medical sciences in 2010 was 47.13 . based on these results , our students had higher levels of happiness which could be due to facilities , the status of their field of study and university , their future career perspectives , and their inner attitudes . on the other hand , the statistical sample size , the age range , and demographic conditions can justify these differences . in our study , secure attachment style was associated with higher happiness scores and this finding was consistent with the findings of other studies . people with secure attachment style are successful in making relationships with others and have positive attitudes about self and others ; the mentioned items are effective in creating higher levels of happiness . researches also show that people with insecure attachment styles are more affected with emotional and psychological challenges and with increasing the feeling of helplessness in the marital relationship , they will be at lower levels of happiness . in a study , girls with secure attachment style , compared with girls with avoidant attachment style , were more satisfied with relationships with their fathers . as another results of our study , there was a significant relationship between happiness scores and gender ; accordingly , the happiness scores in boys was higher than that in girls . in keshavarz et al.s study , contrary to the results of our study , there was a positive relationship between female sex and happiness that could be due to differences in the studied populations . we studied students , while in keshavarz et al.s study , yazd population ( males and females ) were studied . study , no significant relationship was observed between sex and happiness . however , in solymani 's et al . study , men achieved higher scores in subscales of life satisfaction and self - esteem while men had higher scores in a positive manner and inner satisfaction . to interpret these differences , it can be said that working and educational condition , society 's attitudes toward gender , which is strongly influenced by cultural factors , can affect a person 's happiness . in our study , there was a negative correlation between age and happiness scores ; however , this relationship was not significant . in sheikhmoonesi et al.s study the happiness scores in people aged below 22 years were higher than that in people aged more than 22 years . to justify the consistency between the two studies , we can note the similarities in the field of study and age range . in keshavarz et al.s study , older age was associated with greater happiness which could be due to differences in population and age range . in boogar et al.s study , job satisfaction among younger nurses was higher than that in older people . in our studied population , individuals at different ages are not facing the same stressors and expectations ; indeed , the course materials , environmental conditions , and people whom they are communicating with ( professors , personals working in different wards , and patients ) are different at any stage . life satisfaction is not an objective and stable trait , rather it is sensitive to situational changes and is shaped based on individual 's perceptions and perspectives . in multiple regression analysis which was performed with the control of key factors , variables of secure attachment style , gender , and gpa were associated with higher happiness scores . such an analysis has not been carried out in other studies and is one of the strengths of our study . the higher gpa was associated with higher happiness scores and other studies have not addressed this issue . there was higher level of dissatisfaction and expectation among people with lower gpas ; on the other hand , students with higher gpas are dealing with more stress of keeping current situation and they have more competition with others . moreover , mediocre gpa did not indicate higher dissatisfaction , and it might even signify less competitive pressure and family expectations ; this greatly originates from individual 's attitudes and expectations . perfection - seeking individuals may excessively get higher scores , but they are less satisfied and happy . according to our results , the satisfaction score was not significantly associated with educational level which was consistent with the results of sheikhmonesi et al.s study . every educational level brings up different external conditions and stressors which may have different effects depending on the internal characteristics , student 's ability to cope with environment , and individual 's expectation , behavior , and social interaction with others . based on the findings of this study , the most common attachment style was secure attachment style , which could be a positive prognostic factor in medical students , helping them to manage stress . the frequency of avoidant attachment style among single persons was higher than that in married people , which is mainly due to their negative attitude toward others and failure to establish and maintain relationships with others . the variables of secure attachment style , male gender , and average gpa were associated with higher happiness scores these factors can be taken into account while planning for promoting happiness levels in students .

the monthly incidence of listeriosis infections in england and wales had 2 sudden increases during april 2001 ( 41% ) and march 2003 ( 48% ) . although no causative association is demonstrated , these increases correspond to key dates relating to the onset and aftermath of the 2001 foot and mouth disease outbreak in the united kingdom . background : attachment theory is one of the most important achievements of contemporary psychology . role of medical students in the community health is important , so we need to know about the situation of happiness and attachment style in these students.objectives:this study was aimed to assess the relationship between medical students attachment styles and demographic characteristics.materials and methods : this cross - sectional study was conducted on randomly selected students of medical sciences in kurdistan university , in 2012 . to collect data , hazan and shaver 's attachment style measure and the oxford happiness questionnaire were used . the results were analyzed using the spss software version 16 ( ibm , chicago il , usa ) and statistical analysis was performed via t - test , chi - square test , and multiple regression tests.results:secure attachment style was the most common attachment style and the least common was ambivalent attachment style . avoidant attachment style was more common among single persons than married people ( p = 0.03 ) . no significant relationship was observed between attachment style and gender and grade point average of the studied people . the mean happiness score of students was 62.71 . in multivariate analysis , the variables of secure attachment style ( p = 0.001 ) , male gender ( p = 0.005 ) , and scholar achievement ( p = 0.047 ) were associated with higher happiness score.conclusion:the most common attachment style was secure attachment style , which can be a positive prognostic factor in medical students , helping them to manage stress . higher frequency of avoidant attachment style among single persons , compared with married people , is mainly due to their negative attitude toward others and failure to establish and maintain relationships with others .

human acute radiation syndrome ( ars ) follows intense , acute whole - body or significant partial - body radiation , of doses > 1 gy , delivered at relatively high rates . clinical components of ars include the hematopoietic sub - syndrome ( h - ars , 2 6 gy ) , gastrointestinal sub - syndrome ( gis ; 6 8 gy ) and the cerebrovascular ( > 8 gy ) sub - syndrome . dividing ars into these sub - syndromes oversimplifies the clinical reality of ars as it often involves complex , concurrent and multi - organ dysfunctions . cerebrovascular sub - syndrome is considered incurable , whereas h - ars alone or in combination with gis , are more likely to be amenable to countermeasures ; therefore , the later two sub - syndromes are specific targets for the development of novel medical countermeasures ( mcm ) . there are several biologics at different developmental stages to be considered as mcm for ars ( figure 1 , tables 1 and 2 ) . a brief description and current status of promising biologics are provided in this article . schematic representation of the biological agents as radiation countermeasures under development . currently , there are three agents with fda ind status : entolimod , hemamax and neupogen . neupogen and leukine have been procured for sns availability and are expected to obtain fda eua in the near future . pegylated g - csf is not currently stocked in the sns but may also obtain fda eua approval once filgrastim is approved . ars : acute radiation syndrome ; eua : emergency use authorization ; fgf : fibroblast growth factor ; ind : investigational new drug ; sns : strategic national stockpile . ars : acute radiation syndrome ; eua : emergency use authorization ; fgf : fibroblast growth factor ; ind : investigational new drug ; sns : strategic national stockpile . biologics with us fda ind / procured for sns / close to fda approval . gi : gastrointestinal ; ind : investigational new drug ; nhp : nonhuman primate ; pahpra : pandemic and all hazards preparedness reauthorization act ; rhuil-12 : recombinant human il-12 ; sns : strategic national stockpile . we are unable to cite all relevant references because of limitation of total number of references for this article . ars : acute radiation syndrome ; fgf : fibroblast growth factor ; fgf - p : fibroblast growth factor - derived peptide ; gi : gastrointestinal ; gis : gastrointestinal sub - syndrome ; nhp : nonhuman primate . biologics are evaluated for marketing by the fda through the filing of a biologic license application ( bla ) , the equivalent to new drug application for other agents . the agents discussed here qualify for consideration by the fda under bla for approval . since conventional human clinical efficacy trials for ars mcm are not possible due to ethical reasons , these trials are substituted with animal efficacy rule , a very stringent and possibly more difficult fda approval pathway . the criteria required to move through this approval process include : well - characterized animal model(s ) that is predictive of human response , a good understanding of the mechanism of action of radiation injury and that of the mcm , study end point focused on prevention of mortality or major morbidity , as well as the good understanding of pharmacodynamics so that the effective human mcm doses can be determined . this pathway relies heavily on a large animal model for preclinical safety and efficacy studies . the majority of agents at advanced stages of development have received fda fast track and orphan drug statuses . the fda fast track approval process is designed to facilitate development and expedite the review and approval processes for new treatments of serious or life - threatening conditions . radiation mcm for ars are considered emergency need drugs . the us emergency use authorization ( eua ) program , established by project bioshield , is a critical tool for the medical and public health communities . it permits the fda to approve the emergency , off - label use of products approved for other indications or the use of drugs , devices and medical products that currently have no prior approval , clearance or licensing by fda . it is applicable to both civilian and military use , and it fills the need for timely medical treatment in emergency situations . the strategic national stockpile ( sns ) program ensures that such agents are appropriately pre - positioned so that they are readily available and easily accessible for state and local public health agency distribution in the event of a national emergency . as stated above , there are several biologics under development as radiation mcm for ars ( tables 1 and 2 , figure 1 ) . neupogen and two others , entolimod and hemamax , have considerable efficacy and safety profiles and have received fda investigational new drug status for clinical investigation . mechanistic studies have suggested that the various countermeasures for ars have different modes of action ( figure 2 ) . simplified representation of systemic biological effects due to radiation exposure , with promising biologics intervening at various steps . radiation induces free radical formation , dna damage and apoptosis , which can then lead to ars or death . various biologics are able to minimize the damaging effects of irradiation through different mechanisms of action . ars : acute radiation syndrome ; fgf - p : fibroblast growth factor - derived peptide ; mpc : myeloid progenitor cells . ars : acute radiation syndrome ; fgf - p : fibroblast growth factor - derived peptide ; mpc : myeloid progenitor cells . csf have high potential and well - documented therapeutic efficacy in countering myelosuppression and may receive full licensing approval from the fda in the future . filgrastim , sargramostim and pegylated filgrastim have already been used off - label for treating radiation accident victims . currently , there are four recombinant leukocyte growth factors with bla for related indications : bla 103353 ( neupogen ) , bla 125031 ( neulasta ) , bla 103362 ( leukine ) and bla 125294 ( tbo - filgrastim ) . g - csf / filgrastim has completed a good laboratory practice compliant study in a nonhuman primate ( nhp ) model . the use of growth factors to treat radiation - exposed victims has been rationalized based on three facts : i ) a large clinical database documenting consistent efficacy in mitigating chemotherapy - induced myelosuppression and that associated with stem - cell transplant conditioning regimens as well as consistent safety profile ; ii ) enhanced recovery from radiation - induced myelosuppression in four animal species and improved survival in sublethal and lethally irradiated animal models ; and iii ) demonstration of effective granulopoietic activity in a number of radiation - accident victims . additionally , the american society of clinical oncology extended their recommendation for use of recombinant human g - csf ( rhu g - csf ) and pegylated rhu g - csf to treat patients exposed to therapeutic doses of total - body radiation . radiation - accident reports show that csfs have been used to treat the victims of 16 radiological and nuclear accidents with observed benefits . in three accidents csfs were used within 48 h of accidents ( tokai - mura , soreq and nesvizh ) , but in others csf administration was initiated weeks after the incidence . the limited and anecdotal clinical data available regarding these growth factors validate their biological response ; however , the variable and delayed manner in which these agents were administered makes the csf s role in recovery difficult to determine concisely . during a recently conducted fda meeting , members overwhelmingly voted ( 17:1 ) to support that filgrastim will produce clinical benefits to humans who have been exposed to radiation with doses capable of inducing myelosuppression . the one committee member , who voted no , concluded that those who survive a radiological or nuclear incident will most likely not have received a radiation dose high enough to produce myelosuppressive effects and therefore would not benefit g - csf administration . its pharmacological action is based on its binding to toll - like receptor 5 and the activation of nf-b signaling . studies conducted with entolimod using rodent and nhp ( good laboratory practice ) models suggest that it will be a highly promising treatment for lethally irradiated humans , due to its ability to efficiently ameliorate h - ars and gis , as well as having an extended therapeutic time window after radiation exposure [ 6 - 8 ] . entolimod is currently in clinical development ; a human safety study indicated that it was well tolerated and the biomarker results correspond to data from animal models . ( buffalo , ny , usa ) is preparing a pre - eua application for entolimod . hemamax is recombinant human il-12 ( rhuil-12 ) cytokine and has been shown to increase mice survival when a single dose was administered , either 24 h before or within 1 h after total - body irradiation . allometrically equivalent doses of mouse and human hemamax had similar pharmacokinetics and significantly increased mouse and nhp survival , when administered 24 h post - irradiation , even when no antibiotics , fluids or blood products were administered . to demonstrate the safety of hemamax , neumedicines conducted a phase ib study where healthy volunteers were administered a single dose of hemamax that is predicted to be the effective dose for treating h - ars , based on nhp data ; this trial suggests rhuil-12 to be safe and well tolerated . phase ii equivalent data ( randomized , double - blinded , good laboratory practice ) showed that single administration of rhuil-12 to nhps significantly increased survival and reduced radiation - induced hematopoietic toxicity when administered 24 h post - irradiation . administration of rhuil-12 promotes multilineage hematopoietic recovery , immune functions and possibly , gi functions . neumedicines is developing rhuil-12 for the treatment of h - ars for bla submission to the fda under the animal efficacy rule . several additional biologics have been identified as potential countermeasures and have shown promise in murine and nhp models of ars . some of these agents have already been used off - label in radiological accident victims . hopefully , in the future , they will be fully developed agents to combat radiation injury . some of these potential agents are myeloid progenitors , anti - ceramide antibody , fibroblast growth factor-2 ( fgf-2 ) , its derived peptide ( fgf - p ) , igf-1 and various cytokines ( table 2 ) [ 16 - 18 ] . csf have high potential and well - documented therapeutic efficacy in countering myelosuppression and may receive full licensing approval from the fda in the future . filgrastim , sargramostim and pegylated filgrastim have already been used off - label for treating radiation accident victims . currently , there are four recombinant leukocyte growth factors with bla for related indications : bla 103353 ( neupogen ) , bla 125031 ( neulasta ) , bla 103362 ( leukine ) and bla 125294 ( tbo - filgrastim ) . g - csf / filgrastim has completed a good laboratory practice compliant study in a nonhuman primate ( nhp ) model . the use of growth factors to treat radiation - exposed victims has been rationalized based on three facts : i ) a large clinical database documenting consistent efficacy in mitigating chemotherapy - induced myelosuppression and that associated with stem - cell transplant conditioning regimens as well as consistent safety profile ; ii ) enhanced recovery from radiation - induced myelosuppression in four animal species and improved survival in sublethal and lethally irradiated animal models ; and iii ) demonstration of effective granulopoietic activity in a number of radiation - accident victims . additionally , the american society of clinical oncology extended their recommendation for use of recombinant human g - csf ( rhu g - csf ) and pegylated rhu g - csf to treat patients exposed to therapeutic doses of total - body radiation . radiation - accident reports show that csfs have been used to treat the victims of 16 radiological and nuclear accidents with observed benefits . in three accidents csfs were used within 48 h of accidents ( tokai - mura , soreq and nesvizh ) , but in others csf administration was initiated weeks after the incidence . the limited and anecdotal clinical data available regarding these growth factors validate their biological response ; however , the variable and delayed manner in which these agents were administered makes the csf s role in recovery difficult to determine concisely . during a recently conducted fda meeting , members overwhelmingly voted ( 17:1 ) to support that filgrastim will produce clinical benefits to humans who have been exposed to radiation with doses capable of inducing myelosuppression . the one committee member , who voted no , concluded that those who survive a radiological or nuclear incident will most likely not have received a radiation dose high enough to produce myelosuppressive effects and therefore would not benefit g - csf administration . its pharmacological action is based on its binding to toll - like receptor 5 and the activation of nf-b signaling . studies conducted with entolimod using rodent and nhp ( good laboratory practice ) models suggest that it will be a highly promising treatment for lethally irradiated humans , due to its ability to efficiently ameliorate h - ars and gis , as well as having an extended therapeutic time window after radiation exposure [ 6 - 8 ] . entolimod is currently in clinical development ; a human safety study indicated that it was well tolerated and the biomarker results correspond to data from animal models . ( buffalo , ny , usa ) is preparing a pre - eua application for entolimod . hemamax is recombinant human il-12 ( rhuil-12 ) cytokine and has been shown to increase mice survival when a single dose was administered , either 24 h before or within 1 h after total - body irradiation . allometrically equivalent doses of mouse and human hemamax had similar pharmacokinetics and significantly increased mouse and nhp survival , when administered 24 h post - irradiation , even when no antibiotics , fluids or blood products were administered . to demonstrate the safety of hemamax , neumedicines conducted a phase ib study where healthy volunteers were administered a single dose of hemamax that is predicted to be the effective dose for treating h - ars , based on nhp data ; this trial suggests rhuil-12 to be safe and well tolerated . phase ii equivalent data ( randomized , double - blinded , good laboratory practice ) showed that single administration of rhuil-12 to nhps significantly increased survival and reduced radiation - induced hematopoietic toxicity when administered 24 h post - irradiation . administration of rhuil-12 promotes multilineage hematopoietic recovery , immune functions and possibly , gi functions . additionally , there is a report of successful interspecies dose conversion . neumedicines is developing rhuil-12 for the treatment of h - ars for bla submission to the fda under the animal efficacy rule . several additional biologics have been identified as potential countermeasures and have shown promise in murine and nhp models of ars . some of these agents have already been used off - label in radiological accident victims . hopefully , in the future , they will be fully developed agents to combat radiation injury . some of these potential agents are myeloid progenitors , anti - ceramide antibody , fibroblast growth factor-2 ( fgf-2 ) , its derived peptide ( fgf - p ) , igf-1 and various cytokines ( table 2 ) [ 16 - 18 ] . since no fda - approved ars mcm exits , there is an urgent need to develop such agents . based on studies with large numbers of nhps , entolimod appears to be a promising radiation countermeasure for h - ars as well as for gis . entolimod s existing efficacy , safety data and animal - to - human dose conversion are enough to proceed with a pre - eua application to reduce the risk of death following radiation exposure . independent of the fda s approval and licensing process , the us federal government has procured filgrastim and sargramostim to be stockpiled in the sns under the pandemic and all - hazards preparedness reauthorization act . filgrastim and pegylated filgrastim have demonstrated efficacy in recently conducted nhp studies , and these data have been submitted to the study sponsor for submission to the fda for approval . in a recent study , g - csf failed to demonstrate efficacy in the nhp model ; however , this discrepancy may be due to the lack of supportive care . the center for disease control currently holds both investigational new drug and eua applications with the fda for the use of neupogen / g - csf in the event of a nuclear or radiological incident . however , the stability , the implementation plans in the radiological nuclear incident scenario and potential side effects are not fully understood and are of concern . a single injection of hemamax , without supportive care , significantly improved survival and promoted multilineage hematopoietic recovery in an nhp model of h - ars . each drugs sponsor have made clinical progress ; these drugs are moving forward to fill the need for mcm that has increased over the past decade due to increased terrorist threats . in our opinion , the above biologics hold the most promise in the future due to their limited side effects and would be safe and effective agents when approved . neural plasticity refers to the capacity of our brain to change in response to internal demand and/or external experience . burgeoning research has corroborated that the neural plastic changes induced in our brains and behaviors are specific to the experiences ( e.g. , [ 27 ] ) . for instance , the london taxi drivers who have protracted experience in driving around the city with complex road infrastructure demonstrated significant increases in brain structural volume in the posterior hippocampus , which is implicated in storing spatial representation of the environment , suggesting that intensive experiences with spatial navigation can induce specific neural plastic changes in the corresponding brain region . similarly , lee et al . have identified distinct patterns of neural activation associated with different forms of meditation practice , namely , focused - attention meditation ( fam ) and loving - kindness meditation ( lkm ) , during the performance of the sustained attention task . while fam is a form of meditation practice with a heavy emphasis on focusing attention on a particular object , lkm stresses on the cultivation of a state of universal love and compassion as to relieve pain and suffering for the self and others . their findings revealed that fam practitioners ( both experts and novices ) showed significantly greater neural activation in the attention - related network when engaging in the sustained attention task , whereas similar neural activation was not observed in the lkm practitioners . the dissociable neural pattern associated with fam and lkm indicated that meditation , as a form of mental exercise , could induce domain - specific neural plastic changes that accord with the form of meditation practice . these findings offer an insight that perhaps a cognitive experience can be designed to specifically target triggering experience - dependent neural plastic changes in the cognitive domain of interest . yet , our brain , albeit malleable , inevitably undergoes certain degrees of age - associated cortical degeneration at the molecular level as age advances . for instance , significant reduction in global and regional gray matter volume , particularly in the prefrontal and medial temporal regions ( e.g. , [ 1013 ] ) , as well as age - related alterations in the functional connectivity of the default mode network ( e.g. , [ 14 , 15 ] ) have been consistently reported in older adults . substantial research has yielded evidence of age - related decrements in attention and working memory performance ( e.g. , [ 1620 ] ) . mani et al . observed that older adults committed significantly more errors than younger adults when performing a task on sustained attention , suggesting that the ability to sustain attention ( in terms of response accuracy ) was compromised during the natural aging process . it has been speculated that older adults , in order to compensate for their deficits in attentional performance , are required to recruit additional cognitive resources [ 20 , 22 ] . observed that the performance of older relative to younger adults declined with increased working memory load . in terms of long - term memory , profound changes in episodic memory have also been evidenced [ 24 , 25 ] , which can be attributed to the age - associated degeneration in the medial temporal regions , especially the hippocampus . findings from a six - year longitudinal study revealed that hippocampal atrophy and a reduction of neural activation in the hippocampus were only identified in older adults demonstrating declines in memory performance but not in those with intact memory function . research on neuroplasticity - based intervention for age - associated cognitive changes is timely . despite the deleterious effects of age on the brain and behavior , increasing research has shown that the neural plastic potential is preserved until late adulthood ( e.g. , ) . cabeza found that high - performing older adults could counteract age - associated neural decline by recruiting bilateral prefrontal regions when performing a cognitively demanding source memory task , as opposed to the right lateralization observed in both younger adults and low - performing older adults . the reduction in hemispheric asymmetry appears to compensate for the age - associated decline in cognitive performance by reorganizing the neural recruitment [ 28 , 29 ] . in another study , boyke et al . found that older adults demonstrated a similar extent of gray matter changes in the middle temporal area of the visual cortex ( hmt / v5 ) as did their younger counterparts , after acquiring the skill of juggling a three - ball cascade . although the juggling performance of older adults was less proficient than that of their younger counterparts , the observed brain structural changes in the visuomotor region bolstered up the claim that an aging brain is still capable of change in response to experience . further support was substantiated by colcombe et al . , which showed that older adults demonstrated significant increases in brain structural volume after completing aerobic exercise training , in comparison to that of the older adults in the stretching control group . considering that our brain retains its neural plastic potential until late adulthood , lately there has been a growing trend toward exploring any cost - effective intervention that can mitigate or even slow down the age - related declines in cognitive functions . such cognitive training or interventions are primarily targeted at training either general cognitive function or specific cognitive domains , such as attention and working memory , and promising findings have been received thus far ( e.g. , [ 3235 ] ) . building on the principle of neuroplasticity and the concept of the sensory deprivation model , smith et al . developed a self - administered cognitive training program that aimed at improving the auditory processing speed as well as its accuracy . their program consists of six computerized exercises that stress sharpening one 's ability to discriminate , judge , recognize , and match the sequences or pairs of confusable syllables ; to reconstruct the sequences of verbal instruction ; and to discern the details after listening to a story presented verbally . individuals are encouraged to perform cognitive exercises for one hour every day , five days per week , for a total of eight weeks ( equivalent to 40 sessions ) . it was found that participants demonstrated significantly greater improvement in auditory attention and memory after completing the brain fitness program , when compared with their peers in the active control group . recently , the feasibility and potential of implementing the cognitive training program to older adults at risk of cognitive decline have garnered increasing empirical attention and equivocal evidence has been obtained ( e.g. , [ 37 , 38 ] ) . it is important to gather evidence on whether the cognitive training can induce general or specific neural plastic changes in cognitive function among the older adults who experience greater than normal rate of age - related cognitive decline and predispose to greater risk of developing dementia or specifically ad ( e.g. , [ 39 , 40 ] ) . to bridge the aforementioned research gap , the current study was a longitudinal study that examined the specific cognitive effects of planned experience delivered in a cognitive training program . the program , modeled after the brain fitness program , aimed at providing the training for attention and working memory but not verbal or visual - spatial memory of older adults at risk of cognitive decline . based on the principle of experience - induced neural plasticity , it is hypothesized that participants from the cognitive training ( ct ) group , relative to their peers in the active control ( ac ) group , would demonstrate training - specific improvement in attention and working memory but not verbal and visual - spatial memory following 13 weeks of cognitive training . ethics approval of this study was granted by the institutional review board of the university of hong kong / hospital authority hong kong west cluster ( hku / ha hkw irb ) . informed consents for participation in this study were obtained from the 209 right - handed community - dwelling chinese older adults at risk of cognitive decline recruited via a local monthly newsletter , local elderly centers , and word of mouth . these older adults were considered at risk of cognitive decline because their montreal cognitive assessment ( moca ) scores fell into the range of 19 to 26 . other than meeting the moca 's cutoff score , the participants also fulfilled the following inclusion criteria : they ( 1 ) were 60 years old or above , ( 2 ) were literate , ( 3 ) had normal or corrected - to - normal vision and/or hearing , ( 4 ) were right - hand dominant as assessed by the lateral dominance test , and ( 5 ) had normal intelligence as measured by the test of nonverbal intelligence ( toni - iii ) . participants were excluded from this study if they met one of the criteria below : they ( 1 ) had current or a history of neurological or psychological disorders ( e.g. , head injuries , stroke , major depression , or generalized anxiety disorder ) , ( 2 ) had current or a history of substance abuse and/or alcoholism , ( 3 ) were on antidementia medication and/or ( 4 ) were diagnosed with thyroid dysfunction or vitamin b12 deficiency , and ( 5 ) scored in the moderate or severe range ( 11 ) in either subscales ( depression or anxiety ) of hospital anxiety and depression scale ( hads ) . the 209 participants were randomly assigned to the ct and ac groups by an experimenter blind to the cognitive status of the participants using computer - generated random sequences of numbers . specifically , each participant i d was paired with a random number and the order of the participants was rearranged based on the value of the assigned number ( from smallest to largest ) . results of significance test confirmed that the ct and ac groups did not differ in demographic characteristics , cognitive processing speed , and depression scores , variables that have been consistently reported to play a significant role in modulating the rate of cognitive decline ( e.g. , [ 4548 ] ) . the training protocol used in this study was modelled after the brain fitness program ( posit science , san francisco , california , glenn smith ) . it is a self - administered training program of three one - hour sessions per week for a total of thirteen weeks . older participants in the ct group were encouraged to practice four out of six cognitive exercises ( approximately 15 minutes each ) in each training session . in the following , a brief description of each of the six cognitive exercises was provided . in each trial , participants are presented with two sound sweeps . each sound sweep either begins low and rises upward or begins high and goes downward . participants are instructed to indicate the frequency of the two sound sweeps by clicking the corresponding arrows using the mouse . represents that the frequency of the sound sweep shifts from low to high , whereas the down - arrow means that the frequency of the sound sweep shifts from high to low . their task is to discriminate between the different sizes of the heptagons and to identify which heptagon ( left or right ) has a relatively larger size . to increase the level of difficulty , the size contrast decreases ( i.e. , the size difference is not that apparent ) as the number of trials advances . their reaction time and response accuracy for each trial are recorded . in each trial , eight cards are shown on a screen . each card is associated with a syllable . whenever the participants click on each card , their task is to pair up the same syllables that are randomly dispersed among the eight cards . participants can hear the sound as many times as they can but they are encouraged to complete the matching task using the minimal number of steps . their reaction times and response accuracy for each trial are recorded . in each trial , participants hear two pairs of rhythm and are asked to recognize whether the two pairs of rhythm are the same or different . the level of difficulty increases with increased length of the rhythm . the participants ' reaction times and response accuracy for each trial are recorded . in this exercise , participants are first shown three stars appearing in random locations on the screen in quick succession . their task is to repeat the sequence and location where the three stars were shown on the screen in the same order . if the participants ' performance reaches a certain level of accuracy , they would proceed with four - star and five - star conditions . participants are instructed to listen to a number of short narrative stories that comprise short conversations and answer the questions pertaining to the details of the conversation afterward . the purpose of the ac group was to ascertain whether the observed improvement in cognitive functioning can be attributed to factors ( e.g. , the training format ) other than the training content . to account for the confounding factors , the training duration , frequency , and format of the ac group were comparable to those of the ct group , except the content of the training . participants in the ac group did not work on the same kinds of cognitive exercises as did their peers in the ct group . instead , they were shown educational programs covering diverse topics ( e.g. , history , science , health information , and local social issues ) on a group basis . immediately after watching the video , they were instructed to answer several questions that were related to the video content . the current study adopted a longitudinal study design to evaluate the effects of a brain plasticity - based training program on enhancing attention and working memory in a group of chinese older adults at risk of cognitive decline ( figure 1 ) . all participants from the ct and ac groups were required to attend a total of 39 one - hour training sessions over thirteen weeks in groups of four to eight . for both the ct and ac groups , each participant was assigned a laptop , a headset , and a mouse that were used for performing the cognitive exercises . all the training sessions were conducted in a quiet and well - lit room in our laboratory . a research assistant was present in each training session to keep track of their attendance and address any questions pertaining to the task instruction raised by the participants . these research assistants were also responsible for conducting the posttraining assessments . to capture training - associated changes in cognitive function , several cognitive measures on sustained attention , working memory , and memory were administered to all the older adults before and after completing the cognitive training . the digit vigilance test is a measure of vigilance and sustained attention that requires participants to cross out a target number , as fast as they can , throughout the page of digits mixed with other numbers . the numbers are randomly dispersed throughout the page , which adds to the difficulty of identifying the target numbers . both the reaction time and number of errors are recorded . in our study , only the reaction time was included for statistical analysis . the seashore rhythm test taps an individual 's ability to discriminate between similar and dissimilar pairs of rhythms and demands auditory attention . participants listen to one pair of musical beats at a time and are asked to judge whether each pair of musical beats is the digit span test of the wechsler memory scale , third edition ( wms - iii ) , is a measure of auditory working memory . subjects are instructed to repeat the strings of digits in the same ( forward sequence ) and reverse ( backward sequence ) order that is verbally presented by the examiner . the level of difficulty increases as the length of the string of the digits increases . the wms - iii visual - spatial span test assesses the visual - spatial working memory and requires the subjects to touch or point at the blocks in the same ( forward sequence ) and reverse ( backward sequence ) sequences as demonstrated by the examiner . the logical memory subtests of the wms - iii were used to measure verbal memory . subjects are required to recall two stories that are verbally presented by the examiner immediately and after a delay . following the delayed recall trials , a forced - choice yes / no recognition test is also administered . the participants were shown four pictures , one at a time , and were asked to recall the details of each picture . prior to the statistical analyses , an independent t - test was conducted to examine if any between - group differences ( ct and ac ) in the demographic variables ( age , gender composition , and levels of education ) , general intellectual abilities ( scores on toni - iii ) , cognitive processing speed ( processing speed index on the wechsler adult intelligence scale , third edition ( chinese version ) ) , cognitive status ( scores on the cantonese version of moca ) , and depression ( scores on the geriatric depression scale ) were present . a 2 ( training groups : ct and ac ) 2 ( time - points : pretraining and posttraining ) repeated measures anova was used to examine the training effect on sustained attention , working memory , and memory , in chinese older adults at risk of cognitive decline . post hoc comparisons ( paired sample t - tests ) were carried out for each significant interaction effect to further clarify the effect of cognitive training on a specific outcome measure . our final sample consisted of 209 older adults aged 60 to 88 years ( 164 females and 45 males ; age : m = 70.1 years ; sd = 6.38 years ) who successfully completed the pre- and posttraining assessment , of which 109 older adults were randomly assigned to the ct group ( 87 females and 22 males ; age : m = 70.1 years ; sd = 6.21 years ) and 100 older adults were in the ac group ( 77 females and 23 males ; age : m = 70.0 years ; sd = 6.60 years ) ( table 1 ) . participants from the ct and ac groups were matched for their demographic characteristics ( age , gender composition , and years of education ) , cognitive processing speed , and depression scores ( all p > .05 ) . seashore rhythm test and digit vigilance test are the indicators of the auditory and visual attention . a 2 ( training groups : ct and ac ) 2 ( time - points : pre- and posttraining ) repeated measures anova only revealed significant interaction effect on the seashore rhythm test , f(1,207 ) = 5.054 , p = .026 , but not on the reaction time of the digit vigilance test , f(1,207 ) = .046 , p = .831 ( table 2 ) . post hoc comparison indicated that only the ct group demonstrated significant improvement in the seashore rhythm test following the training , t(108 ) = 3.707 , p < .000 , whereas the ac group did not show any significant change in their scores but simply maintained a similar level of performance on the seashore rhythm test upon completion of the training , t(99 ) = .796 , p = .428 . digit and visual - spatial span assess the verbal and visual - spatial attention and working memory , respectively . the two - way repeated measures anova only identified significant interaction effects on the total digit span , f(1,207 ) = 6.473 , p = .012 , as well as the total visual - spatial span , f(1,207 ) = 5.047 , p = .026 , which represent auditory and visual - spatial attention and working memory ( table 2 ) . post hoc comparisons showed that the ct group exhibited significant increases in the total digit span , t(108 ) = 4.119 , p = .000 , and total visual - spatial span , t(108 ) = 3.835 , p = .000 , after three months of cognitive training . in contrast , the ac group did not show any statistically significant increase or decrease in the total digit span , t(99 ) = .121 , p = .904 , and total visual - spatial span , t(99 ) = .552 , p = .582 . both immediate and delayed recall trials of wms - iii subtests of logical memory and family picture neither the main effects nor interaction effects on the immediate and delayed recall trials of wms - iii logical memory and family picture were significant ( all p > .05 ) ( table 2 ) . to explore whether levels of cognitive status would have been associated with different training outcomes , pearson correlational analyses were carried out between the baseline general cognitive statuses ( as measured by moca ) and the pre- versus posttraining changes in cognitive measures among the older participants from the ct group . none of the pre- versus posttraining changes in cognitive variables ( including attention , working memory , and cognitive processing speed ) was significantly correlated with the initial general cognitive statuses of the participants from the ct group ( all p > .05 ) . this study used a longitudinal design to examine the specific effect of planned experience on cognitive functioning . consistent with our a priori hypothesis , behavioral changes are specific to the types of experience induced to the brain . in this study , only attention and working memory , but not verbal and visual - spatial memory , showed an improvement after the 13-week systematic cognitive training of these cognitive domains . this pattern of finding is consistent with the prediction set forth by experience - dependent neural plasticity model . the specific improvement in attention and working memory can not be attributed to other demographic , cognitive , or emotional variables because the ct and ac groups were matched on these dimensions . these training - induced modality - specific improvements in attention and working memory performance have underscored the importance of designing a tailor - made experience as to induce experience - specific changes in cognitive functions ( e.g. , [ 27 , 49 , 50 ] ) . perhaps future research can consider exploring the potential of cognitive intervention that is targeted at training a specific cognitive domain or even a specific modality . for example , it is well - documented that atrophy of the medial temporal lobe ( especially in the hippocampus ) may explain the aging - related memory impairment [ 2426 ] . based on the findings of this study , planned cognitive training inducing learning and memory consolidation may lead to experience - specific neural plastic changes in the hippocampus leading to long - term memory enhancement . furthermore , while memory is the major cognitive domain being affected in people suffering from alzheimer 's disease , training targeted at verbal and visual - spatial memory may be beneficial in slowing the memory decline . the nonsignificant training effect on sustained attention , measured by the digit vigilance test , is inconsistent with previous findings . we speculated that the discrepant findings could relate to the use of different age cohorts between ours and previous studies . literature has pointed out that normal aging is associated with widespread neuronal and synaptic atrophy and physiological degradation , which contribute to age - related blunting of neuroplastic responses in an aging brain . on the ct group , our correlational analyses showed that their initial general cognitive status was not significantly associated with any of the pre- versus posttraining changes in cognitive variables of interests ( including attention , working memory , and memory ) , indicating that older adults who were considered to be at greater risk of cognitive decline could capitalize on the cognitive training to a similar extent and their initial cognitive status may not confine the neural plastic potential of their aging brains . considering that our study specifically targeted older adults who scored within the restricted range of moca ( i.e. , 19 to 26 ) , it remains unclear whether the significant correlation identified in our study can be generalized to the elderly population who scored in the normal range , so it is worthwhile to look into this unaddressed question in the future . the findings of this study could be complemented by neuroimaging data that can inform the neural changes associated with the observed behavioral improvement . future research could investigate whether cognitive training can trigger changes at both the behavioral and neural levels , which could definitely advance our understanding of the mechanisms underlying the training effects . future studies can probe into the temporal window during which the transition from the neural plastic changes to behavioral changes can be captured . previous reports have suggested the importance of understanding the generalization of the training effect to activities of daily living ( adl ) and other untrained cognitive domains . unfortunately , we were unable to test for the generalization of the training effects beyond the laboratory setting . it is worthwhile to devote the research effort to elucidate the mechanism underlying the training - transfer effects in order to augment the beneficial effect of cognitive training brought to older adults . while the current study did not intend to inspect the potential impact of educational background on the training effect and hence matched for older participants ' level of education , our previous study did report that the less educated older adults were more likely to gain from the cognitive training than their better educated peers . future investigation can gauge the potential role of educational background and possibly other factors as to maximize the extent to which the older adults can take advantage of the cognitive training . cortical degeneration accompanied by aging , coupled with cognitive deterioration , has placed a heavy socioeconomic burden on the health care system . increasing research has evidenced that our brain retains a capacity to change in response to experience until late adulthood . our findings shed insight into the potential of implementing cognitive training for older adults at risk of cognitive decline and provided substantial support that the neural plastic potential continues until older age . most importantly , this study has provided strong evidence for the potential application of the experience - induced neuroplasticity model to develop cost - effective strategies that can potentially slow down the rate of cognitive decline associated with aging .

despite significant scientific advances toward the development of a safe , nontoxic and effective radiation countermeasure for acute radiation syndrome ( ars ) over the past six decades , no drug has been approved by the us fda . several biologics are currently under development as radiation countermeasures for ars , of which three have received fda investigational new drug ( ind ) status for clinical investigation . presently , two of these agents , entolimod ( cblb502 ) and hemamax ( recombinant human il-12 ) are progressing with large animal studies and clinical trials . neupogen ( g - csf , filgrastim ) has recently been recommended for approval by an fda advisory committee . filgrastim , gm - csf ( leukine , sargramostim ) , and pegylated g - csf ( neulasta ) have high potential and well - documented therapeutic effects in countering myelosuppression and may receive full licensing approval by the fda in the future . the former two biologics are available in the us strategic national stockpile ( sns ) for use in the event of nuclear or radiological emergency . the emergency use authorization ( eau ) application for entolimod may be filed soon with the fda . biologics are attractive agents that are progressing along the path for fda approval , to fill the unmet need for ars countermeasures . increasing research has evidenced that our brain retains a capacity to change in response to experience until late adulthood . this implies that cognitive training can possibly ameliorate age - associated cognitive decline by inducing training - specific neural plastic changes at both neural and behavioral levels . this longitudinal study examined the behavioral effects of a systematic thirteen - week cognitive training program on attention and working memory of older adults who were at risk of cognitive decline . these older adults were randomly assigned to the cognitive training group ( n = 109 ) and the active control group ( n = 100 ) . findings clearly indicated that training induced improvement in auditory and visual - spatial attention and working memory . the training effect was specific to the experience provided because no significant difference in verbal and visual - spatial memory between the two groups was observed . this pattern of findings is consistent with the prediction and the principle of experience - dependent neuroplasticity . findings of our study provided further support to the notion that the neural plastic potential continues until older age . the baseline cognitive status did not correlate with pre- versus posttraining changes to any cognitive variables studied , suggesting that the initial cognitive status may not limit the neuroplastic potential of the brain at an old age .

chemically , phloroglucinol ( 1,3,5-trihydroxybenzene , pg ) and its methylated derivative tri - o - methylphloroglucinol ( tmp ) , figure 1 , are established pharmaceutical agents inhibit the action of catechol - o - methyl transferase , inducing relaxation of smooth muscles , and decreasing glycerol - induced abdominal pain and are also characterized by a swift and strong spasmolytic activity , hence relieving pain . therefore , pg is often used in combination with trimethylphloroglucinol as an antispasmodic drug and is regarded to be effective in decreasing smooth muscle spasm . pg / tmp combination is recommended against biliary calculi , severe pain of urinary or gastrointestinal tract , pain of abdominal region , spastic conditions of the female genital system , and pain in gynecology [ 17 ] . literature survey reveals that some of the analytical methods for phloroglucinol are available including extraction and high - performance liquid chromatography ( hplc ) [ 810 ] , hplc - mass spectrometry [ 11 , 12 ] , gas chromatography - mass spectrometry , and spectrophotometry . other reported methods include titrimetry , spectrophotometry , paper chromatography , and flow injection analysis [ 1520 ] . however , there is no simple and sensitive method to be followed on industrial basis especially in general quality control laboratories . therefore , they can not be followed in the laboratories particularly those of third world countries . hence our group already developed a cost effective method for its fixed dose composition in tablet form , but still there was a need for an analytical method which would help to determine the active pharmaceutical ingredients ( apis ) in parenteral products and physiological fluid . accordingly , the purpose of this write - up is to suggest a systematic approach for the development of a validated simple , sensitive , and stability indicating rp - hplc method that should meet the current ich and regulatory requirements . the present method was designed to be easy to use , sensitive , and rapid . separation and quantification of pg and tmp in pharmaceutical drug formulations and blood were achieved with an isocratic elution and with dual wavelength technique . sil 10a autoinjector hplc system comprising of scl 10a system , controller , spd 20a prominence uv / vis detector , and shimadzu lc 20 at pump with lc solutions software , was used . separation was performed on a hibar bondapak ods c18 hplc column ( 4.6 250 mm ; 10 m bead size ) and maintained at 25c . a uv - visible shimadzu 1650 pc spectrophotometer with uv probe software , ultrasonic cleaner ( elmasoni e 60 h ) , jenway 3240 ph meter , and sartorius te2145 analytical balance were used in the research work . throughout the work only amber glass flasks were used to avoid light effect on the solution of pg and tmp standards and samples . trimethylphloroglucinol and phloroglucinol were kind gifts from a national pharmaceutical company , and sulphuric acid ( merck , germany ) and acetonitrile ( hplc grade ) were purchased from fisher scientific . the injectable containing pg and tmp were obtained from commercial source ( spasfon injection , spadix injection , furosinol injection , and anafortan plus injection ) labeled to contain 10 mg / ml and 0.01 mg / ml of pg and tmp , respectively . the compound was chromatographed isocratically with a mobile phase consisting of acetonitrile ( hplc grade ) : sodium n - heptane sulphonate ( 0.005 m ) : sulfuric acid 0.1 m ( 50 : 50 : 0.3 , v / v / v ) with the ph adjusted if required to 3.0 0.1 using 0.1 m sulfuric acid or 0.1 m naoh . while for sample and standard preparation diluent was prepared from acetonitrile ( hplc grade ) : distilled water ( 50 : 50 , v / v% ) . the mobile phase was filtered by passing through a 0.45 m membrane filter ( millipore , bedford , ma , usa ) and degassed via sonication . the flow rate was 1.0 ml / min , and the injected volume was 20 l . the effluent was monitored at dual wavelength of 266 and 205 nm . in a 100 ml volumetric flask , about 10 mg of tmp reference standard was weighed accurately and dissolved in diluent to have a stock solution of 100 g / ml . similarly in another 100 ml volumetric flask , accurately 500 mg of pg reference standard was weighed and dissolved in diluent , sonicated for 2 minutes , and let to cool to room temperature . then , 5 ml of tmp stock solution was added , and volume was made up to the mark with the same diluent . finally 2 ml of this solution was diluted in 100 ml of diluent to get a 100 g / ml of pg and 0.1 g / ml of tmp working standard solution . the standard was then filtered through 0.45 m filter paper and injected into the hplc system . for making a sample of 100 g / ml of pg and 0.1 g / ml of tmp , 10 ampoules were broken , and content was mixed to get an evenly homogenized stock sample . the sample volume was taken accurately equivalent to 10 mg of pg & 0.01 mg of tmp in 100 ml volumetric flask , and 50 ml of diluent was added . the sample was sonicated for 1 minute and then diluent was added up to the mark and placed on stirrer for 5 minutes . the sample was then filtered through 0.45 m filter paper and injected into the hplc system . blood samples were collected from healthy volunteers in evacuated glass tube through an indwelling cannula placed on forearm vein by a trained clinical laboratory technician . the volunteers were not involved in any medication , smoking , and strenuous activity . the blood was shaken and centrifuged at 6,000 rpm for 30 min to separate out plasma . 9 ml acetonitrile was added to 1 ml plasma and centrifuged at 6,000 rpm for 30 min to deproteinate it . the supernatant serum thus obtained was stored at 20c and filtered for subsequent analysis . for making a working sample solution of 12.5 ml , stock sample was taken in 50 ml flask followed by 30 ml of serum addition . the sample thus obtained was stirred for 10 minutes and then diluent was added up to the mark . further 2 ml of this solution was diluted in 50 ml of flask with the aid of diluent . all samples prepared were filtered through 0.45 m membrane filter and injected in triplicate into the hplc system . for this purpose 5 ml of the stock sample was diluted in 100 ml diluent to make a stock sample solution . for working purpose , 10 ml of stock sample solution was diluted in four individual 50 ml volumetric flasks , and 15 ml of degrading agent were added to each flask , with the exception of one to which only diluent was added ; these included 0.1 n hcl , 0.1 n naoh , and 30% h2o2 , and then to each flask diluent was added up to the mark . the samples were then filtered through 0.45 m membrane filter and injected into the hplc system . intentional degradation was attempted with stress conditions exposing the drugs to acid ( 0.1 n hcl ) , alkali ( 0.1 n naoh ) , hydrogen peroxide ( 30% ) , and heat ( 60c ) to evaluate the ability of the proposed method to separate drugs from its degradation products . for all conditions the temperature was kept constant at 60c for a period of one hour . for stability studies the commercially available injection ( parenteral ) samples in ampoules were placed at accelerated conditions of temperature that is at 40c with 75% relative humidity and at ambient conditions of 30c temperature with 65% relative humidity in environmental chamber for six months . the stability protocol in table 6 various procedures including specificity , linearity , range , accuracy , and intraday and interday precision were evaluated . to study linearity , twenty dilutions of standard were made to prepare standard solution in range from 10% to 200% , that is , from 10 g / ml to 200 g / ml of pg and from 0.01 g / ml to 0.2 g / ml of tmp , respectively , drugs content . the standard calibration curve was generated using regression analysis with online help ( http://wessa.net/ ) . for specificity commonly used excipients in injection preparation were spiked in a preweighed quantity of drugs to determine the effect and interference of excipients in quantification of the drugs . in order to find out the repeatability and reproducibility of the method , precision was studied to find out intra- and interday variations in the test method of pg and tmp in the concentration range of 80120 g / ml for pg and 0.080.12 g / ml for tmp , respectively . precision was determined by analyzing corresponding bulk sample daily for a period of three days and three times a day with an interval of 8 hours against a freshly prepared standard . for determining accuracy the pg and tmp reference standard were accurately weighed and spiked to the injection sample at three different concentration levels to gain 110% , 120% , and 130% of both apis . at each level , limit of detection and quantification ( lod and loq ) for the method were established by sequentially diluting the standard solutions at decreasing concentrations , in the range of 1001 pg / ml for pg and 101 pg / ml for tmp . the limit of detection was defined as the concentration for which a signal - to - noise ratio of 3 was obtained , and for quantification limit , a signal - to - noise ratio of 10 was considered . the standards were injected in lc system , and measured signals from the diluted standards were compared with those of blank samples . to study robustness , samples of injections were assayed with deliberate variation in the method parameters , such as in the chromatographic conditions , like mobile phase , flow rate , and temperature so forth . justification of system suitability was established by calculating % relative standard deviation of replicate injections and analyzing the symmetry , resolution of the standard peaks , and theoretical plates of the column . the hplc method development and its validation are the prioritized requirements for any drug available in the market to ensure the quality of the products . a few methods are available for determination of the apis as described earlier , but many of them are used only for certain definite objectives and lack generalization for simultaneous analytical applications in form of pharmaceutical products and serum . similarly none of them are as much sensitive as ours in terms of their precision , accuracy , limit of detection ( lod ) , and limit of quantification ( loq ) especially as compared to [ 7 , 9 , 12 , 14 ] whose lod or loq was in microgram range only , while ours method is sensitive enough to be used for pharmacokinetic studies . here , the lod and loq for both apis are 10 pg / ml and 33 pg / ml , respectively ; however considering their ratio in sample formulation , the lod and loq for pg are taken as 10 ng / ml and 33 ng / ml , respectively , and hence loq and lod for tmp can also be considered as the levels of sensitivity of the method . for developing an efficient method for the simultaneous analysis of pg and tmp , parameters such as detection wavelength , mobile phase composition , optimum ph , and concentration of the standard solutions both pg and tmp were diluted in dilution solvent and then run through uv spectrophotometer in uv range of 190 nm400 nm to get maximal wavelengths , figure 2 , where maximum absorbance was gained , that is , 266 nm for both apis . however considering the difference of concentration in both apis that is 10 to 0.01 mg / ml for pg and tmp , respectively , therefore 205 nm was used for tmp and 266 nm for pg . the chromatographic parameters were evaluated using a hibar bondapak ods c18 column the mobile phase composed of acetonitrile : buffer of given proportion promoted a short run time ( 10 min ) without any interference , so this condition was adopted in subsequent analysis . the literature survey also revealed that almost all the methods developed so far have utilized acetonitrile as a major component in mobile phases . acetonitrile is always preferred due to the supreme solubility properties and uv absorbance characteristics , and there is no counterpart substitute for acetonitrile in the reverse phase hplc and uv application . therefore , keeping in view the chromatography type and the detection wavelengths in use , acetonitrile was chosen for analysis , though in our previous work we used methanol for its cost effectiveness . the linearity ranges were found to be 10200 g / ml for pg and 0.010.2 g / ml for tmp . the assay was judged to be linear , as the correlation coefficient was 0.9999 and 0.9992 for pg and tmp , respectively , as calculated by the least - square method . a linear correlation was found between the peak areas and the concentrations of apis , in the assayed range . chromatogram shown in figure 3 proves specificity or selectivity of the assayed method , as the chromatograms in samples were found identical with standard chromatogram and no interference peak was observed in sample chromatogram . peak purities higher than 98.0% were obtained in the chromatograms of sample solutions , demonstrating that other compounds did not coelute with the major peaks . the chromatogram obtained with the mixture of the injection excipients proves that there is no interference from excipients and peak of interest that fulfills all the requirements of symmetrical peak , and hence , the specificity is confirmed . the precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple sampling of homogeneous bulk . intraday precision of the method was evaluated at three different independent concentrations that are , 80% , 100% , and 120% for both drugs ( n = 3 ) by determining their assays . interday precision of the method was tested for 3 days at the same concentration levels . since the interday and intraday precision obtained % rsd were less than 2% it is assured that the proposed method is quite precise and reproducible , as shown in table 2 . the accuracy was investigated by spiking reference standards to a mixture of the injection excipients at three different concentration levels , that is , multiple level recovery studies , and subjected to the proposed hplc method . the obtained recovery ( n = 9 ) was 98.86100.96% ( rsd% = 0.488 ) for pg and 98.56%101.24% ( rsd% = 0.139 ) for tmp , demonstrating the accuracy of the method . percentage recoveries for marketed products were found to be within the limits , table 3 . the statistical analysis showed no significant difference between results obtained by employing the analytical conditions established for the method and those obtained in the experiments in which variations of some parameters were introduced . the parameters used in system suitability test were symmetry of peaks , tailing factor , resolution , and rsd% of peak area for replicate injections . thus , the method showed to be robust for changes in mobile phase acetonitrile proportion , mobile phase ph , flow rate , and column temperature , table 4 . during the degradation study , it was observed that upon treatment of pg and tmp with base ( 0.1 m naoh ) , acid ( 0.1 m hcl ) , hydrogen peroxide ( 30% ) , and heat , maximum degradation was observed in acid and h2o2 . it is a fact that phenolic compounds , such as phloroglucinol , are known to undergo ready oxidation in basic solutions ( due to dissolved oxygen ) and by agents such as hydrogen peroxide which increases the absorbance capacity of the molecules , causing increased peak area and hence % calculated amount . and the same is revealed by the data given that pg is more vulnerable to stress conditions especially alkaline and oxidation treatments as compared to tmp . degraded peaks are observed in case of acid hydrolysis , and peak had been broadened due to extra absorbance caused by oxidation of the pg , whereas no appreciable degradation was observed in heat treated sample ; hence the drug is stable under heat stress conditions . the purpose of stability testing is to provide evidence of how the quality of a drug substance or drug product varies with time under a variety of environmental conditions , like temperature , humidity , and light and enable recommendation of storage conditions , retest periods , and shelf life to be established . the two main aspects of drug product that play an important role in shelf - life determination are assay of the active drug and the degradation products generated during stability studies . the proposed assay method was applied to the stability study of commercially available injections , for which the samples were placed at 30c with relative humidity of 65% and at 40c with relative humidity of 75% . according to the results obtained both apis were found to be stable at applied conditions of temperature and relative humidity and were accurately analyzed with the proposed method . the proposed new hplc method described in this paper provides a simple , universal , convenient , and reproducible approach for the simultaneous identification and quantification of phloroglucinol and trimethylphloroglucinol in human serum and pharmaceutical formulations ( injectable ) with good separation and resolution . in addition , this method has the potential application to clinical research of drug combination . analytical results are accurate and precise with good recovery and lowest detection limit values . in short , the developed method is simple , sensitive , easy , and efficient having short chromatographic time and can be used for routine analysis in qc laboratory and therapeutic monitoring . fibromyalgia ( fm ) , which affects at least 10 million american adults , is a multisymptom condition resulting in not only widespread chronic pain , but also fatigue , sleep disturbances , and morning stiffness . in addition , many patients experience depression , anxiety , and dyscognition [ 2 , 3 ] . fm has a significant adverse impact on many individuals ' physical and mental health [ 4 , 5 ] and also leads to reduced workplace productivity and increased health care / disability expenses , with the estimated cost of fm on the us economy being reported to be 1214 billion dollars [ 1 , 6 ] . due to the lack of clarity for the underlying cause(s ) of fm , it poses a diagnostic challenge , often requiring multiple visits by specialists to render a diagnosis . the lack of understanding of the biological basis of this condition also confounds our ability to develop effective interventions and/or monitor disease progression . fm has been suggested to be a complex , multifactorial trait that is influenced by age , gender ( frequency is the highest in middle - aged females ) , and stress / trauma . despite showing a strong familial aggregation [ 810 ] , attempts to identify genetic factors associated with fm ( primarily through polymorphism association studies ) have yielded inconsistent results , with some investigators showing associations between fm and specific genes ( including , but not limited to , genes for catechol - o - methyltransferase [ 1113 ] , serotonin-2a receptor [ 14 , 15 ] , serotonin transporter gene regulatory region [ 16 , 17 ] , dopamine d4 receptor , -2 adrenergic receptor , gamma - aminobutyric acid receptor subunit beta-3 , trace amine - associated receptor 1 , interferon - induced guanylate - binding protein 1 , regulator of g protein signaling 4 , cannabinoid receptor type 1 , and glutamate receptor 4 ) , while others failed to identify a relationship [ 2125 ] . since a consistent , straightforward association with a gene(s ) has not yet been forthcoming , scientists have suggested that the familial influence on fm may more likely reflect a genetic susceptibility to environmental events [ 21 , 26 , 27 ] . for example , klengel and binder identified differential methylation for a glucocorticoid response element ( the fkbp5 gene ) that resulted from the presence of both an at - risk allele ( polymorphism ) and the occurrence of childhood trauma in subjects they studied who had posttraumatic stress disorder . epigenetics , which refers to the process that affects gene expression independent of actual dna sequence ( such as methylation changes , histone alterations , and micro - rna expression ) , has enabled scientists to conceptualize the impact of the environment upon one 's genes and one 's health . genes can be turned on or off and display variations in their level of expression , in part , due to epigenetic modifications . thus , epigenetics provides a biological means for understanding the molecular processes of complex biological networks that connect the brain , behavior , and health outcomes . given the overlap in symptoms and the medical / adverse social histories present in people who have fm , when compared to other conditions that have been shown to be impacted by somatic epigenetic and genetic alterations ( such as depression and stress ) , it is plausible that similar epigenetic mechanisms may underlie the individual variability in the outcome of genetic and emotional inputs for fm . knowing that histone and other epigenetic modifications play a key role in establishing and maintaining chromatin structure , it follows that changes in epigenetic profiles , as a consequence of initiating events ( such as stress / environmental exposure ) , could also lead to an increased frequency of somatic chromosomal changes . indeed , we have shown that stress levels can impact the frequency of acquired chromosomal abnormalities by demonstrating a significantly increased frequency of somatic cell chromosomal instability in adult women who experienced childhood sexual abuse when compared to their identical cotwins who did not experience childhood sexual abuse ( quantified using a micronucleus assay ) . further support that chromosomal instability could arise as a downstream effect following perturbations in methylation comes from studies of individuals who have immunodeficiency , centromeric region instability , and facial anomalies ( icf ) syndrome , which is an autosomal recessive condition resulting from a mutation in the methyltransferase gene b. people with this condition have an increased frequency of acquired chromosomal abnormalities . an efficient means for quantifying the frequency of acquired ( somatic ) chromosomal abnormalities is the cytokinesis block micronucleus ( cbmn ) assay , which provides information regarding the presence of chromosomal errors in somatic cells with minimal influences attributable to in vitro selective growth pressures . this technique is less labor intensive than conventional cytogenetic studies but provides results that are in close agreement to those obtained using the gold standard of metaphase chromosomal analyses . micronuclei , which are the primary cytological structures scored in the cbmn assay , are thought to contain chromatin ( from one or more chromosomes ) that was not incorporated ( lagging or lost ) into the daughter binucleates following nuclear division . micronuclei frequencies have been shown to increase with age and have been associated with a variety of health conditions [ 36 , 37 ] . however , to date , no investigators have reported the frequency of mn in women with fm . based on the symptomatology and comorbidities related to this condition , we hypothesized that women with fm will have an increased frequency of acquired epigenetic and chromosomal alterations . to test this hypothesis , we initiated a pilot study to quantify chromosomal instability levels and genome - wide methylation patterns in women having fm and to compare these genetic / epigenetic values to those present in comparably aged , healthy control women . data for this study were obtained from a subset of participants ( n = 10 ) , who were randomly recruited by mail from a larger , parent study sample of 64 women diagnosed with fibromyalgia ( vcu irb protocol number hm12211 ) ( table 1 ) . in the parent study , the participants completed a two - group randomized , controlled , clinical trial to examine the effect of guided imagery on self - efficacy , perceived stress , pain , fatigue , depression , and neuroendocrine / neuroimmune biomarkers in women with fibromyalgia syndrome . inclusion criteria for the women having fm were age ( at least 18 years old ) ; gender ( female ) ; receipt of a physician - confirmed diagnosis of fm based on the 1990 american college of rheumatology criteria ; an ability to understand and sign the consent form ; and an ability to understand / complete the study questionnaires . exclusion criteria for the women in the fm pilot group included the presence of other systemic rheumatologic conditions ; being immunocompromised ( e.g. , diagnosis of hiv / aids ) ; receiving corticosteroid treatments ; having a major psychiatric or neurological condition that would interfere with study participation , or being pregnant . each of the study subjects completed a self - report form to provide information regarding age , race / ethnicity , marital status , length of time since diagnosis of fibromyalgia , height and weight for calculating body mass index ( bmi ) , and lifestyle practices such as history of smoking and alcohol use . the healthy , comparatively aged control group of women for the mn studies ( n = 42 ) were ascertained through their participation in a study of acquired genetic changes associated with normal aging , the latter of which is a twin study ( vcu irb protocol number 179 ) . the inclusion criteria for this subset of control subjects were gender ( female ) and age ( range from 36 to 69 years old ) , with all people from the previous study who met the criteria being included as controls for the current study to avoid sampling biases . the control cohort of women included both cotwin pairs ( n = 30 women or 15 cotwin pairs ) and single twins , whose cotwin did not participate in this normal aging study ( n = 12 women ) . due to cost limitations , dna methylation studies were limited to a subset ( n = 8) of the control women . this subset of women was randomly selected and included 8 unrelated females ( no cotwins ) . all of the control women self - reported their age , race / ethnicity , and lifestyle practices , such as history of smoking and alcohol use . following the collection of the blood specimens from the patient and control women , the cells were processed to obtain binucleates for the micronuclei studies and dna for the methylation studies as described in the following section . lymphocytes were collected using histopaque-1077 ( sigma ) , stimulated with the mitogen phytohemagglutinin , established in culture , and blocked at cytokinesis according to the protocol of fenech . briefly , cytochalasin b ( 3.0 g / ml ; sigma , 14930 - 92 - 2 ) was added to the cells 44 hours after culture initiation . at 72 hours , binucleate interphase cells were harvested using standard techniques , which included a 10-minute incubation in hypotonic solution ( 0.075 m kcl ) and serial fixation ( three times using a 3 : 1 methanol : acetic acid solution ) . micronuclei were visualized following giemsa staining ( 4% harleco giemsa solution ) and identified according to the criteria established by fenech ( figure 1 ) . the frequencies of micronuclei observed in the cytochalasin - b - blocked binucleated cells of the women with fm and the healthy control women were calculated by averaging the values obtained from two replicate scores ( 1000 binucleates were evaluated from two independent areas of the slide ) for a total of 2000 binucleates that were evaluated per study participant . genomic dna was isolated from whole blood according to standard methods using the puregene dna isolation kit ( qiagen ) . an aliquot ( 500 ng per study participant ) of dna was then sent to hudson alpha institute for biotechnology for bisulfite conversion , using standard methods ( zymo research ez methylation kit ) and genome - wide methylation pattern assessment , using the 450 k humanmethylation chip , according to the manufacturer 's protocol ( illumina ) . intensity data from the scanned arrays were imported into illumina 's genomestudio software and the minfi bioconductor package in the r programming environment to obtain the values for each probe . to test the hypothesis that women with fm have an increased frequency of acquired chromosomal alterations , the frequency of mn given that a portion of the healthy controls were cotwins , a mixed effect model was used to control for familial relationships . mn frequency comparisons between cases and controls were adjusted for age since several studies have demonstrated increases in mn frequency with age [ 37 , 4042 ] . additional environmental effects were considered that have previously been shown to influence micronuclei frequencies . these included body mass index , alcohol use , and smoking in the women having fm . however , given that values for body mass index , alcohol use , and smoking were not available for the controls ( in a format comparable to those for cases ) , these variables were only assessed for the women with fm . because the 450 k humanmethylation assay includes both the infinium i design ( which includes two probes for interrogating a cpg site ) and the infinium ii design ( which includes only one probe ) , the gc content was plotted separately by infinium design type for the fully methylated sample , for which all cpg sites are expected to have consistently high values . based on these results , probes having a gc content greater than 40 were excluded from further analysis to ensure that the results would not be biased by the in addition , since the performance of probes containing single nucleotide polymorphisms ( snps ) can be variable , probes containing snps that were present within 10 bases of the target site were also excluded . because the expression value , , reported for each cpg site represents proportion methylated which is constrained to an interval value from 0 to 1 , where a of 1 indicates complete methylation and 0 indicates no methylation , the expression values were transformed using the logit transformation [ m = log(/(1 ) ] to promote normality . prior to the logit transformation , imputation was completed ( 0.001 for values that were 0 and 0.999 for values that were 1 ) to avoid nonexistent m values . to adjust for the observation that values from the infinium ii designed probes have a compressed range compared to the values from the infinium i design [ 43 , 45 , 46 ] , the peak - correction method was applied to the logit transformed values for the infinium ii designed probes . statistical analyses were then performed on the peak corrected logit transformed values from the patient and control samples . for each cpg site , differential methylation between specimens collected from women with fm and controls was tested using the moderated t - test in the limma bioconductor package [ 51 , 52 ] in the r programming environment . to adjust for the multiple hypothesis tests , the p values were used to estimate the false discovery rate ( fdr ) following benjamini and hochberg 's method . the david gene functional classification tool was used to identify biological relationships among the differentially methylated sites . as expected , the frequency of mn was correlated with age in both the women having fm ( r = 0.717 ; p = 0.021 ) and the healthy controls ( r = 0.579 ; p = 4.79 10 ) ( figure 1 ) . after controlling for the effect of age and cotwin status , a significantly increased frequency of mn was observed in the women having fm ( 51.4 ( 21.9 ) ( mean ( sd ) ) per 1000 binucleates ) compared to controls ( 15.8 ( 8.5 ) ( mean ( sd ) ) per 1000 binucleates ) ( = 45.6 ; df = 1 ; p = 1.49 10 ) ( figure 1 ) . the increased levels of mn in the women having fm were not significantly correlated with their body mass index ( range from 19.44 to 45.70 ; mean ( sd ) was 29.52 ( 7.21 ) ; p = 0.997 ) , smoking history ( 4 smokers ; 6 nonsmokers ; p = 0.75 ) , or alcohol use ( 6 consumers ; 4 nonconsumers ; p = 0.93 ) . to assess if there might be a cumulative biological effect associated with experiencing symptoms associated with fm , we compared mn frequencies for the case subjects ( n = 10 ) with the total number of years that had lapsed since these women received their diagnosis of fm . while there was a trend toward a positive correlation between a woman 's mn frequency and the number of years since she was diagnosed with fm ( ranged from 2 to 19 years ) , this relationship did not reach significance in this small pilot study ( p = 0.134 ) ( table 1 ) . after completion of the quality control assessments that were performed to remove any potential biases associated with probe sequence length , probe gc content , and inclusion of snps , a total of 381,989 cpg sites were retained . from these , a total of 69 sites were determined to be differentially methylated ( dm ) between the patients who have fm and the healthy controls , with 63 of these dm sites having higher values in the patients with fm and 6 having lower values ( figure 2 ) . these 69 dm sites included cpg islands ( 46.4% ) ; north shores ( 20.3% ) ; south shores ( 8.7% ) ; as well as north ( 4.3% ) and south ( 1.4% ) shelves and sites that were not annotated into the previously noted categories ( 18.8% ) . the dm sites were localized to 47 different genes ( table 2 ) , with 3 genes having multiple sites identified ( n - acetyltransferase 15 gene ( nat15 ) had 4 dm sites ; dnaj ( hsp40 ) homolog , subfamily c , member 17 ( dnajc17 ) had 2 dm sites ; and slc17a9 and 2 dm sites ) . an assessment of potential biologically related clusters of dm sites resulted in the recognition of 15 groups , including gene clusters involved in neuron differentiation and nervous system development ( table 3 ) . while the sample size in this pilot study is small , the mn frequency patterns of both the case and control women showed an age - related increase , which is a finding that is in agreement with the age - related increase that has consistently been reported in larger studies [ 40 , 41 ] . interestingly , the mean frequency of mn in the women with fm was 3.26-fold higher than the level seen in the healthy controls . in comparison , patients who have cancer have been noted to have 1.37- to 3.13-fold higher frequencies of mn when compared to healthy controls [ 57 , 58 ] . given that the risk for cancer has been shown to be predicted by mn levels [ 37 , 41 , 58 ] , the results of this preliminary data , if confirmed , suggest that mn frequency assessments may be useful for evaluating / diagnosing women with fm . indeed , recent assessments of mn frequencies in people evaluated from various areas of biobehavioral science have shown increased levels of acquired chromosomal instability ( assessed using mn frequencies ) in adult women who experienced childhood sexual abuse ; patients who have neurodegenerative conditions , such as alzheimer 's disease and parkinson 's disease ; and adults with type 2 diabetes and cardiovascular disease . the presence of acquired chromosomal instability , which could lead to somatic tissue mosaicism , has been conjectured to occur as a global biological process that affects many tissues and contributes to the development of several conditions , including ( but not limited to ) autism , schizophrenia , autoimmune diseases , and alzheimer 's disease . given that several of these conditions are age related , one could speculate that there may be a factors contributing to mn formation are multifold and include both genetic and environmental influences [ 37 , 40 , 41 ] . environmental exposures that have been shown to increase the frequency of mn include , but are not limited to , diet ( especially folate deficiency ) [ 41 , 62 , 63 ] , hormone levels , and exposures to substances / occupational hazards . the biological means whereby these genetic / environmental influences lead to acquired chromosomal instability are likely to be varied but have been noted to reflect the chromatin conformation of the chromosomes . one can speculate that alterations in chromatin conformation , which are likely to arise ( at least in part ) from epigenetic changes , may compromise the ability of the chromosomes to align , attach to mitotic spindle fibers , and/or separate , thereby leading to their increased frequency of abnormalities . in turn , the presence of acquired chromosomal abnormalities could lead to additional epigenetic alterations . while limited in number , studies performed to assess the effect of methylation on chromosome segregation have consistently shown an increase in the frequency of cytogenic abnormalities associated with perturbations in the methylation status of chromosomes . in this study , it is interesting to note that dm sites were identified for genes having a function related to chromatin compaction and/or segregation ( nat15 ; hdac4 ; uhrf1 ) . for example , dm sites were observed for the nat15 gene , which is a gene that has been identified to play an important role in normal chromosomal segregation during anaphase . while the results of the genome - wide methylation patterns evaluated in this study are preliminary , it is exciting to note that several of the sites that were dm between the women with fm and controls were localized to genes that have functional relevance to the symptoms seen in patients with fm . of particular interest was the observation of a significant difference in the methylation pattern of the bdnf gene between patients with fm and controls . the bdnf gene has been noted to play an important neuromodulatory role in pain transduction ( eliciting hyperalgesia ) [ 6971 ] and has also been recognized as a contributor to learning and memory [ 72 , 73 ] . a second gene of apparent relevance with which a dm site was associated was the protein kinase c , alpha gene ( prkca ) ( table 3 ) . this gene , which is involved in cell signaling pathways , has been associated with emotional memory formation , posttraumatic stress syndrome , cancer , and aging . a third gene of particular interest that had a dm site is reticulon 1 ( rtn1 ) . rtn1 has been associated with neurological diseases ( and cancer ) and is thought to influence membrane trafficking in neuroendocrine cells . overall , genes with which dm sites were associated include ( but are not limited to ) those having functions in chromatin compaction ( nat15 ; hdac4 ; uhrf1 ) ; dna damage / repair or chromosomal segregation ( sod3 ; uhrf1 ; nat15 ) ; muscle contraction ( nr4a3 ; hdac4 ; fez1 ; prkg1 ; kcnh7 ) ; axonal bundling / outgrowth ( fez1 ) ; cell signaling in muscle ( nr4a3 ; prkg1 ) ; neuronal excitability / synaptic transmission ( bdnf ; bzrap1 ; epha5 ; kcnh7 ) ; muscle maturation ( hdac4 ) ; response to oxidative stress ( sod3 ) ; and inflammatory processes ( axl ; sh2b2 ) . however , since two of the significant biological clusters that were identified ( table 3 ) were for polymorphisms and sequence variants , it is important to recognize that this is a pilot study and that a larger number of individuals will need to be evaluated to allow one to determine the extent , if consistently present , of dm on the development or severity of symptoms associated with fm . the results of genome - wide methylation studies have provided insight regarding the role of genes and environmental influences for a variety of conditions , with many of these investigations focusing on the areas of cancer and psychiatric conditions [ 74 , 75 ] . however , the epigenomes of diseases causing chronic pain have been less extensively evaluated . in their study of rheumatoid arthritis , nakano et al . observed several dm sites between patients who have rheumatoid arthritis and controls . they also identified distinct epigenomic signatures when comparing patterns from patients with rheumatoid arthritis and osteoarthritis . akin to the results of this pilot study , the findings of their investigation led to the recognition of perturbations in the methylation status of several genes having functions related to the symptoms associated with rheumatoid arthritis . thus , the use of genome - wide epigenetic assessment seems to be a promising tool for evaluating a broad spectrum of conditions , including those associated with chronic pain . in summary , the results of this pilot study suggest that chromosomal instability and alterations in methylation are present in women with fm . if these results can be confirmed , they could provide a basis for improving our understanding of the biological changes leading to the development of fm and may provide a basis for stratifying patients based on their epigenomic and symptom patterns . moreover , since epigenetic changes demonstrate plasticity , the recognition of consistent epigenetic alterations associated with fm could provide a means for developing future therapeutic approaches to reverse these changes , with a goal of alleviating symptoms in people who have fm .

a reverse phase stability indicating hplc method for simultaneous determination of two antispasmodic drugs in pharmaceutical parenteral dosage forms ( injectable ) and in serum has been developed and validated . mobile phase ingredients consist of acetonitrile : buffer : sulfuric acid 0.1 m ( 50 : 50 : 0.3 v / v / v ) , at flow rate 1.0 ml / min using a hibar bondapak ods c18 column monitored at dual wavelength of 266 nm and 205 nm for phloroglucinol and trimethylphloroglucinol , respectively . the drugs were subjected to stress conditions of hydrolysis ( oxidation , base , acid , and thermal degradation ) . oxidation degraded the molecule drastically while there was not so much significant effect of other stress conditions . the calibration curve was linear with a correlation coefficient of 0.9999 and 0.9992 for pg and tmp , respectively . the drug recoveries fall in the range of 98.56% and 101.24% with 10 pg / ml and 33 pg / ml limit of detection and limit of quantification for both phloroglucinol and trimethylphloroglucinol . the method was validated in accordance with ich guidelines and was applied successfully to quantify the amount of trimethylphloroglucinol and phloroglucinol in bulk , injectable form and physiological fluid . forced degradation studies proved the stability indicating abilities of the method . fibromyalgia ( fm ) , characterized by chronic widespread pain , fatigue , and cognitive / mood disturbances , leads to reduced workplace productivity and increased healthcare expenses . to determine if acquired epigenetic / genetic changes are associated with fm , we compared the frequency of spontaneously occurring micronuclei ( mn ) and genome - wide methylation patterns in women with fm ( n = 10 ) to those seen in comparably aged healthy controls ( n = 42 ( mn ) ; n = 8 ( methylation ) ) . the mean ( sd ) mn frequency of women with fm ( 51.4 ( 21.9 ) ) was significantly higher than that of controls ( 15.8 ( 8.5 ) ) ( 2 = 45.552 ; df = 1 ; p = 1.49 1011 ) . significant differences ( n = 69 sites ) in methylation patterns were observed between cases and controls considering a 5% false discovery rate . the majority of differentially methylated ( dm ) sites ( 91% ) were attributable to increased values in the women with fm . the dm sites included significant biological clusters involved in neuron differentiation / nervous system development , skeletal / organ system development , and chromatin compaction . genes associated with dm sites whose function has particular relevance to fm included bdnf , nat15 , hdac4 , prkca , rtn1 , and prkg1 . results support the need for future research to further examine the potential role of epigenetic and acquired chromosomal alterations as a possible biological mechanism underlying fm .

plx4032 was synthesized using the general procedures previously described.6 expression and purification of b - raf , structure determination , protein kinase activity measurements , and xenograft studies were carried out as previously described.6 clinical methods have also been recently described.5 melanoma patients were selected for study using previously described taqman methodology.8 semi - quantitative immunohistochemistry for perk and ki67 was performed on 5 m - thick formalin - fixed paraffin - embedded tumor biopsies following h&e staining to determine pathologic diagnosis and tissue morphology and integrity . the degree of phospho - erk staining in the nucleus and cytoplasm was interpreted semiquantitatively by assessing the intensity and extent of staining on the slides . for ki67 staining , the localization and subsequent translation of mrna are important means of regulating gene expression with high spatial and temporal control . in neurons , targeting mrna to specific sites and synthesizing proteins where and when they are needed is particularly beneficial because neurons have long dendrites and axons extending from a few hundred microns to up to a meter from the cell body . the physiological roles of mrna localization in neurons have been implicated in various neuronal functions such as synaptic plasticity , axonal guidance and regeneration . however , there are many remaining questions regarding how mrnas navigate through the complex neuronal arbors , how they are captured in specific regions , and how local translation is regulated ( buxbaum et al . , 2015 ; holt and schuman , 2013 ; hutten et al . , 2014 ; xing and bassell , 2013 ) . observing the dynamics of mrna localization and translation will provide critical information to help understand how precise spatio - temporal regulation of gene expression is processed . most previous studies have used in situ hybridization ( ish ) methods ( lawrence and singer , 1985 ) to visualize rna molecules inside a cell . in particular , fluorescence in situ hybridization ( fish ) enables the detection of individual rna molecules with various signal amplification procedures in fixed cells . single - molecule fish ( smfish ) , which uses multiple fluorescent probes hybridized to a single mrna ( femino et al . , 1998 ; , 2008 ) , is widely used to profile transcription , localization and degradation of rna . however , fish experiments using fixed cells can not provide temporal information on rna regulation . recent innovations in live - cell imaging technologies have made it possible to observe the sequence of molecular events in real time , which is critical to our understanding of mrna dynamics ( moon et al . , 2016 ; spille and kubitscheck , 2015 ) . by imaging single mrna molecules in live cells , we can begin to understand the cause - and - effect relationship and to model the kinetics of rna regulation more quantitatively and predictably . in this review , we provide a brief overview of recent progress in studies on mrna localization and translation in neurons . in particular , we highlight several recent reports that have used single - molecule imaging techniques to contribute to our knowledge of mrna translocation in dendrites and axons . additionally , we review new technical developments for multicolor imaging of single mrnas and their translational activities . these approaches will provide a powerful toolkit to help understand the molecular mechanisms of rna localization and local translation in neurons , with unprecedented temporal and spatial resolution . since the discovery of polysomes at the base of dendritic spines ( steward and levy , 1982 ) , many lines of evidence have indicated that de novo protein synthesis in dendrites is required for long - term synaptic plasticity ( hanus and schuman , 2013 ; jung et al . , 2014 ) . deep rna sequencing revealed 2,550 mrnas that are present in dendrites and axons in the hippocampus ( cajigas et al . , 2012 ) . a large fraction of these mrnas encode synaptic proteins such as signaling molecules , scaffolds and receptors . among these mrnas , activity - regulated cytoskeleton - associated protein ( arc ) , the -subunit of calcium / calmodulin - dependent protein kinase ii ( camkii ) and -actin mrnas have been extensively studied to unravel their regulation in live neurons . to understand how these mrnas are sorted and transported to dendrites , it is crucial to track the movement of individual mrna molecules ( fig . single - mrna tracking has revealed the diverse and stochastic nature of mrna trafficking ( park et al . , 2010 ) . transport and localization of mrna depend on the interplay of cis - acting rna elements , rna binding proteins ( rbps ) , and motor proteins ( czaplinski , 2014 ) . various kinds of rbps bind to their target mrnas to form messenger ribonucleoprotein complexes ( mrnps ) . after recruiting motor proteins , mrnps are transported along cytoskeletal tracks . a cis - acting rna sequence that confers dendritic localization , which is called a dendritic targeting element ( dte ) , is typically located in the 3 untranslated region ( 3 utr ) of the mrna . , 2000 ) or the arc 3 utr ( dynes and steward , 2007 ; 2012 ) exhibit bidirectional transport in dendrites and localize at the base of dendritic spines . a 54-nucleotide - long zipcode in the -actin 3 utr ( kislauskis et al . , 1994 ) and zipcode binding protein 1 ( zbp1 ) are required for the localization of -actin mrna in dendritic filopodia and axonal growth cones ( eom et al . , 2003 ; zhang et al . , 2001 ) . microinjected camkii and septin7 mrnas colocalize in the same particles , while map2 mrnas reside in different particles , suggesting that some rnas that share a common dte can be sorted in the same complexes and transported to distal dendrites ( tubing et al . , for example , there have been conflicting results for defining the dte in the camkii 3 utr ( doyle and kiebler , 2011 ) . the cis - acting rna elements interact with a set of transacting factors or rbps to form mrnp complexes that are transported along microtubules by kinesin or dynein . tracking reporter rnas in fmr1 knock - out ( ko ) neurons revealed the important roles of fragile x mental retardation protein ( fmrp ) as an adaptor that links mrna to the kinesin light chain ( dictenberg et al . , 2008 ) and for mrna delivery to dendritic spines in response to synaptic stimulation ( kao et al . , 2010 ) . live imaging of neurons also showed that huntingtin ( htt ) , huntingtin - associated protein 1 ( hap1 ) , kif5a , and dynein intermediate chain are associated with -actin mrna during dendritic transport ( ma et al . , 2011 ) . knocking down the levels of these proteins reduced the transport of -actin mrna , suggesting that they have roles in mrna localization in neurons . an actin - based motor , myosin - va , contributes to the transport of tls ( translocated in liposarcoma)-containing mrnps in dendritic spines ( yoshimura et al . , 2006 ) . most previous mrna tracking studies have been performed using transfection or injection of exogenous reporter mrnas . however , reporter rnas usually do not contain all of the cisregulatory sequences and trans - acting rbps and thus may not represent the true characteristics of endogenous rnas . to label endogenous mrna , genetically engineered mice have been generated by inserting ms2 binding site ( mbs ) stemloops into a target rna and expressing ms2 capsid protein ( mcp)-gfp proteins ( lionnet et al . all the endogenous -actin mrnas were labeled with the ms2-gfp system in live mice ( park et al . , 2014 ) . tracking the labeled endogenous -actin mrnas revealed that mrna diffusion was much slower in neurons than in fibroblasts . while most of the -actin mrnas in fibroblasts showed diffusive motion , a small fraction of the -actin mrnas in neurons showed bidirectional motion with a mean speed of 1.3 m / s . recently , glutamate uncaging was used to demonstrate that -actin mrnas can localize in the stimulated dendritic region within 15 min of stimulation ( yoon et al . , 2016 ) . nmda receptors and zbp1 have important roles in the localization process . in neurons cultured from zbp1 ko mice , -actin mrnas were transported to the stimulated region ; however , they did not maintain their localization . the authors also showed that newly synthesized -actin proteins accumulated in the stimulated spine head and that this helped stabilize the expanded spines . taken together , tracking single - mrna movements enables us to reveal how rna transport and localization happen in live neurons . the current working model is called the sushi belt model , in which mrnps stroll along axons and dendrites like sushi plates on a conveyor belt to serve potential customers ( doyle and kiebler 2011 ) . those mrnas may be captured by synaptic tags that are relevant to the formation of long - term potentiation ( ltp ) and long - term depression ( ltd ) ( frey and morris , 1997 ) . further studies using new developments in genetic engineering and in systems biology approaches will elucidate the precise regulatory mechanisms of mrna localization in dendrites . while local translation in dendrites is well accepted , local protein synthesis in axons has been controversial over the years . although a few early studies reported the presence of ribosomes in axons ( bunge , 1973 ; tennyson , 1970 ; zelena , 1970 ) , the low density of axonal mrnas and ribosomes supported the traditional view that axonal proteins are synthesized in the cell body . later , however , two studies showed evidence for local translation in the axons . campbell and holt observed that growth cones of retinal ganglion neurons lose their ability to turn away from a source of sema3a when translation is inhibited ( campbell and holt , 2001 ) . zhang and poo showed that localized synaptic potentiation by bdnf requires presynaptic protein translation in xenopus nerve - muscle cultures ( zhang and poo , 2002 ) . since then , there have been many studies on mrna localization in axons and on the implications of those localized mrnas for axon guidance , survival and regeneration ( gumy et al . , 2014 ; jung et al . , bdnf- or netrin-1-induced growth cone turning is mediated by local translation of -actin mrna ( leung et al . , 2006 ; bdnf induces src - mediated zbp1 phosphorylation , which releases zbp1 from -actin mrna ( huttelmaier et al . , 2005 ) . after zbp1 is dissociated , -actin proteins are locally synthesized from the mrna and then direct growth cone turning . in a similar mechanism , map1b mrnas are also locally translated in response to sema3a ( li et al . , 2010 ) . it has been suggested that fmrp has a role in protein synthesis - dependent collapse of growth cones induced by sema3a ( fig . in addition , branching of sensory axons requires local protein synthesis and the stalling of mitochondria ( spillane et al . , 2013 ) . using a gfp reporter construct containing the 3 utr of -actin mrna , the authors found that hot spots of axonal translation occur at the base of branches that also contained mitochondria . zbp1 is colocalized with myosin - va in axons , and inhibition of myosin - va function increases the number of motile zbp1 particles ( nalavadi et al . , 2012 ) . furthermore , some rbps can bind to several species of mrnas . in dorsal root ganglion neurons , sfpq , a splicing factor , works as an rbp binding to lmnb2 , bcl2l2 , impa1 and creb1 mrnas ( cosker et al . , 2016 ) . in addition , sfpq plays a role in the axonal transport of these mrnas to promote axon survival . in central nervous system ( cns ) axons , studies on mrna localization have been facilitated by the use of microfluidic devices that separate axons from cell bodies and dendrites ( taylor et al . , 2005 ) . using a microfluidic device in conjunction with microarray analysis and fish , many species of mrnas have been detected in the axons of cns neurons as well as of peripheral nervous system ( pns ) neurons . studies of mature cortical axons have identified mrnas that are involved in the translation machinery , transport , cytoskeletal components and mitochondrial maintenance ( taylor et al . , 2009 ) . to date , there are only a few studies on live - cell imaging of axonal mrna transport . using the ms2-gfp system , bi et al . demonstrated that the 5 and 3 utrs of kor ( -opioid receptor ) mrna are required for its axonal transport in dorsal root ganglion ( drg ) neurons ( bi et al . , 2006 ) . more recently , a molecular beacon method was used to visualize nefl ( neurofilament - l ) mrna in axons of primary mouse cortical neurons and human motor neurons derived from ips cells ( alami et al . simultaneous imaging of nefl mrna and trans - active response dna - binding protein 43 ( tdp-43 ) , an rbp related to amyotrophic lateral sclerosis ( als ) , revealed that tdp-43 promotes anterograde transport of nefl mrnp granules . this result suggests that tdp-43 has a role in the delivery of target mrna to distal axons . with the development of microfluidic devices , rna labeling techniques , and high - resolution live - cell imaging , it is now more feasible to study mrna transport in axons . for instance , integrated microfluidic platforms are developed for investigating regeneration of injured axons ( kim et al . , neurons cultured from mcp mbs knock - in mice now allow us to image single endogenous mrnas without transfection steps ( park et al . , 2014 ) . because local translation plays a crucial role in axon development and regeneration , it is important to understand the molecular mechanisms that govern mrna trafficking and localization in axons . translation is a highly regulated , complex biological process in which mrna is decoded by a ribosome to produce a protein . several whole - genome techniques for studying translation have been developed based on rna sequencing ( rna - seq ) or mass spectrometry ( ms ) . ribosome profiling , for example , measures ribosome - protected regions of mrnas with next - generation sequencing ( ngs ) , providing position information on translating ribosomes throughout the cellular transcriptome ( ingolia et al . , 2009 ) . global , averaged measurements of translation initiation , elongation , and termination rates can be made by ribosome profiling ( ingolia et al . , 2011 ) . another sequencingbased technique called translating ribosome affinity purification ( trap ) ( heiman et al . , 2008 ) or ribotag ( sanz et al . , 2009 ) relies on pulling down gfp- or ha - tagged ribosomal proteins and then the identification of mrnas associated with them . ms - based methods such as pulsed stable isotope labeling by amino acids in cell culture ( silac ) ( schwanhausser et al . , 2009 ) and biorthogonal noncanonical amino acid tagging ( boncat ) ( dieterich et al . , 2006 ) have been used to identify newly synthesized proteins after adding labeled amino acids . because genome - wide methods can only detect the average behavior of many cells , imaging - based techniques have been developed to study the dynamics of translation in single cells ( chao et al . , 2012 ) . fluorescent proteins have been used to detect local protein synthesis ( aakalu et al . , 2001 ) ; however , the long maturation time of fluorescent proteins has been a major limitation of such methods . as an attempt to visualize translation from single mrnas , halstead et al . developed an rna biosensor termed translating rna imaging by coat protein knock - off ( trick ) that can distinguish untranslated mrnas from those that have undergone at least one round of translation ( halstead et al . orthogonal bacteriophage pp7 and ms2 stem - loops were used to label mrna with two different fluorescent proteins ( fig . . simultaneous expression of the pp7 capsid protein fused with green fluorescent protein ( pcp - gfp ) and the ms2 capsid protein fused with red fluorescent protein ( mcp - rfp ) resulted in yellow fluorescent signals from untranslated mrnas . during the first round of translation , pcp - gfp is removed from the mrna as a ribosome traverses the coding region that contains the pp7 stemloops . thus , translated mrnas are labeled only with mcp - rfp bound to the ms2 stem - loops in the 3 utr . while the trick technique provides information on the first round of mrna translation , recent studies by four independent groups ( morisaki et al . , 2016 ; wang et al . , 2016 ; wu et al . , 2016 ; yan et al . , 2016 ) have demonstrated two - color imaging of mrna and its nascent polypeptides . these four studies used the ms2 or pp7 systems to label mrna and the suntag ( tanenbaum et al . , 2014 ) or flag tag ( viswanathan et al . , 2015 ) fluorescence - tagged ms2 or pp7 capsid proteins were expressed in cells , providing a strong fluorescence signal from each mrna . the suntag and flag tag systems use fluorescently labeled antibody fragments that bind to multiple copies of a short epitope on each polypeptide . an mrna undergoing active translation can be identified by the colocalization of two fluorescent signals from the mrna and nascent proteins . wu et al . , 2016 ) used the suntag - ms2 technique not only in cell lines but also in hippocampal neurons . they used ornithine decarboxylase ( odc ) or auxin - induced degron ( aid ) to facilitate rapid degradation of the epitope array after translation . this allowed them to distinguish nascent polypeptides translated on the mrna from the background of freely diffusing polypeptides in the cytosol . wang et al . found that the arc 3 utr increased the number of translation sites in the dendrites of neurons . wu et al . showed that ~40% of the reporter mrnas were translated in the proximal dendrites ; however , only ~10% were translated in the distal dendrites . both studies this result contradicts the previous speculation that translation is repressed during the transport of mrna to localization sites . although these findings were obtained using exogenous reporters , the tagging method could be applied to endogenous genes with crispr / cas9 genome - editing techniques ( nelles et al . , 2016 ) . if we could track endogenous mrna while measuring its translational output in response to diverse signaling in live tissues , we would be able to uncover many interesting details regarding the dynamics of gene expression . further improvements in these imaging techniques ( pichon et al . , 2016 ) will certainly offer new insights into the complex regulation of translation at the single - molecule level in situ . the elongated morphology of neurons requires directed transport of mrna and local translation of proteins for efficient gene expression in dendrites and axons . because local protein synthesis has important roles in neuronal development and synaptic plasticity , it is crucial to understand how specific mrna molecules are targeted to the activated regions for local translation . recent advances in live - cell single - rna imaging techniques have significantly increased our knowledge on the dynamics of rna in neurons what are the cis - regulatory rna sequences for selective transport into dendrites and axons ? how are mrnps delivered and captured at the stimulated synapses ? understanding the molecular mechanisms behind rna localization , translation and degradation in neurons will provide clearer answers to questions about the physiological relationships between gene expression and brain functions .

b - raf is the most frequently mutated protein kinase in human cancers.1 the finding that oncogenic mutations in braf are common in melanoma2 followed by the demonstration that these tumors are dependent on the raf / mek / erk pathway3 offered hope that inhibition of b - raf kinase activity could benefit melanoma patients . herein , we describe the structure - guided discovery of plx4032 ( rg7204 ) , a potent inhibitor of oncogenic b - raf kinase activity . preclinical experiments demonstrated that plx4032 selectively blocked the raf / mek / erk pathway in braf mutant cells and caused regression of braf mutant xenografts.4 toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity , enabling phase 1 clinical trials using a crystalline formulation of plx4032.5 in a subset of melanoma patients , pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment . this analysis revealed substantial inhibition of erk phosphorylation , yet clinical evaluation did not show tumor regressions . at higher drug exposures afforded by a new amorphous drug formulation,4,5 greater than 80% inhibition of erk phosphorylation in the tumors of patients correlated with clinical response . indeed , the phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily.5 these data demonstrate that braf - mutant melanomas are highly dependent on b - raf kinase activity . local protein synthesis mediates precise spatio - temporal regulation of gene expression for neuronal functions such as long - term plasticity , axon guidance and regeneration . to reveal the underlying mechanisms of local translation , it is crucial to understand mrna transport , localization and translation in live neurons . among various techniques for mrna analysis , fluorescence microscopy has been widely used as the most direct method to study localization of mrna . live - cell imaging of single rna molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein ( mrnp ) complexes in neurons . here , we review recent advances in the study of mrna localization and translation in live neurons using novel techniques for single - rna imaging .

the modern world is an interconnected mesh of networks satisfying a myriad of functions : transportation , electrical power , food distribution , finance , and health care to name a few . the interoperability of these networks developed as part of urban evolution over the past century such that these and other webs connect to national and/or global networks ( national research council of the national academies , 2005 ) . this is the engineered webbing of humanity , but there are comparable natural structures in the spheres of biology , ecology , sociology , and physiology . this modernity is manifest in the military through the development of network - centric warfare ( ncw ) which takes cognizance of human behavior in a networked environment of organized actions directed toward political , social , and military ends and is the basis of a new theory of war ( office of force transformation , 2004 ; garstka and alberts , unpublished ) . thus , ncw has at its core a shift in focus from military platforms such as ships and tanks to networks having platforms as members . army scientists need to understand the dynamics , controllability , and predictability of generic non - linear complex networks in order to realize their goal of supporting both society and the soldier through research and the development of new technologies . it is not only our external world that is cluttered with networks , but our internal world as well . the neuronal network carrying the brain s signal to the body s physiological networks is even more complex than the modern city or a typical ecological network . thus , the basic research into network science must span and encompass a multitude of disciplines ; understanding each sheds light on the others . the problem addressed within this program is to develop the basic research foundation of a science of networks that supports the linkage between the cognitive and social domains as they relate to decision making . this approach is not directed at the totality of developing a network science , but has the more modest goal of understanding the deeply interdependent human networks of crucial importance to society as a whole and to the army in particular . even such a restricted problem is a significant challenge due to the multiply interconnecting networks buttressing the common decision making objective . on the one hand , the military is proactive in that networked forces can operate in an agile manner to promote decision making superiority . on the other hand , the army is reactive in the need to respond to enemies who are also using the power of networks against united states interests . the research program provides insight to allow the army to anticipate the enemy s use of network strategy and thereby reduce the reactive mode of operation . in grigolini and west ( 2011 ) we reviewed what is presently known about complex networks , regardless of the disciplinary context and adapted that understanding to the decision making paradigm . moreover , the barriers to further understanding and to filling the gaps in knowledge of the linkages between social and human decision making networks were addressed . the research strategy of the arl program is based on theory , computation / simulation , and experiment / observation . this is a cyclic interactive process in which new theory stimulates unique simulations , yielding insight into parameter values and network configurations , which in turn suggests specific experiments , whose outcome guides the refinement and development of theory . this modern approach to scientific research is applied to the phenomenon of human decision making with a view for eventual application to ncw . the core group of army scientists is the focal point for external researchers requiring militarily relevant challenges and internal arl efforts . one of the mysteries of human social interaction is how agreements are reached and cooperative alliances are made . individuals become part of social groups or networks in a number of ways : choice , peer pressure , and subliminal seduction ; but always through a sequence of decisions , either conscious or not . network characteristics can not be deduced from the properties of individuals ; they emerge during the formation and growth of the network . consequently we need mathematics to quantify the strength of the interactions between the network components , as well as to describe how a network develops in time and responds to perturbations ( stimulation ) . this has been done through the construction of the decision making model ( dmm ) that for very weak coupling is much like the ising model of cooperative behavior , but for strong coupling can be very different ( turalska et al . , 2009 ) ubiquitous aspects of complex networks are the appearance of non - stationary , non - ergodic , and renewal statistical processes . law statistical distributions that not only challenge traditional understanding of complexity in physical networks , but require new strategies for understanding how information is exchanged between networks ( west et al . , 2008 ; west and grigolini , 2011 ) , as in the case of interest here among human networks including cognitive networks . the approach is to adapt the methods of non - equilibrium statistical physics that have been used to characterize the dynamics of complex phenomena and phase transitions . these methods were extended to the study of such social phenomena as linguistics , biofeedback techniques , and the brain s response to music ( bianco et al . , 2007 ) and to further develop them to model decision making with incomplete information in an uncertain environment . the mathematics of complex networks has been examined using the newly developed dmm to understand consensus ( turalska et al . , 2009 ) ; a psychophysical model of how individuals make decision and then irrationally change their minds shows agreement between theory and experiments ( west and grigolini , 2010a ) ; renewal statistics reveal how we habituate to the familiar ( west and grigolini , 2010b ) and forget the uninteresting ( west and grigolini , 2010c ) ; 1/f variability captures how the brain processes tasks of increasing complexity during decision making ( grigolini et al . , 2009 ) ; and finally we have determined how all these various pieces fit into the overall picture of exchanging information between complex networks ( west et al . , 2008 ; grigolini and west , 2011 ; west and grigolini , 2011 ) . the major accomplishment of the present research program has been the identification of the first universal principle in the science of networks , that being , the principle of complexity management ( pcm ) discussed in grigolini and west ( 2011 ) , west and grigolini ( 2011 ) . pcm states that the maximum information is exchanged between two complex networks when there is compatibility of the complexity of the two networks . a mathematical proof of this principle has been constructed over the past year ( aquino et al . , 2010 , 2011 ) . the mathematician norbert wiener speculated that the transfer of influence from a complex network high in information to one low in information even though the latter may be higher in energy represents a new kind of interaction ( wiener , 1985 ) , which we called wiener s rule ( grigolini and west , 2011 ) . his insight was vindicated a half century later ( aquino et al . , 2010 , 2011 ) and required the generalization of a number of concepts from statistical physics ( allegrini et al . , 2007 , 2011 ; aquino et al . , 2007 ; budini and grigolini , 2009 ) resulting in the pcm ( west et al . , 2008 ; west and grigolini , 2011 ) as we discussed last year ( grigolini and west , 2011 ) . one measure of the information content of a network is provided by the probability density most often used in the determination of the negative entropy of shannon and wiener . an apparently ubiquitous distribution in the description of empirical complex networks is the hyperbolic , having the survival probability : which asymptotically becomes an inverse power law . the average time between events in complex webs such as power grid blackouts , heartbeats , time between earthquakes ( west and grigolini , 2011 ) of a given magnitude can be determined using the probability density (t ) = d(t)/dt , to be it is interesting that when the power < 3 the distribution has a finite first moment and the statistics are ergodic , meaning that the time average and ensemble averages yield the same result . law index is < 2 there are no finite integer moments and the time and ensemble averages are not the same , that is , the process is non - ergodic . one measure of the information transfer between two complex networks is the cross - correlation between a complex network p and a complex network s being perturbed by p with the strength of the perturbation . for our purposes it is sufficient to apply the generalized linear response theory ( lrt ; aquino et al . , 2010 , 2011 ) we previously developed the normalized cross - correlation function : the perturbing complex network p is characterized by the non - stationary autocorrelation function p ( t , t ) , which depends separately on the time of the last perturbation t and time of the measurement t. the function rs(t ) is the rate of generating perturbing events at time t within the network being perturbed and is based on renewal theory ( west and grigolini , 2011 ) . the perturbed network s is characterized by the stationary autocorrelation function s ( t t ) , which depends only on the difference in times from the last perturbation to the measurement . in figure 1 the asymptotic cross - correlation function normalized to the strength of the perturbation is graphed as a function of the power the cube displays a number of remarkable properties : ( 1 ) when the power law indices are both equal to two there is an abrupt jump from zero correlation in region ii to perfect consensus in region iii ; ( 2 ) the upper plateau region iii indicates that when p is non - ergodic 1 < p < 2 and s is ergodic 2 < s < 3 there is an information response in which the perturbed network tracks the perturbing network exactly and the information transfer is maximal ; ( 3 ) when p is ergodic 2 < p < 3 and s is non - ergodic 1 < s < 2 there is no response asymptotically and the information transfer is minimal as shown in region ii . how a complex network responds to a perturbation by another complex network is determined by the kind of mismatch that exists in the complexity of the fluctuations in the two networks . the cross - correlation cube . the asymptotic cross - correlation function defined by eq . law indices of the perturbed network s and the perturbing network p. ( from west and grigolini , 2011 , with permission . ) wiener s rule describes the influence of the perturbing network outside the lower plateau region of the cross - correlation cube . in all regions except this one the weak perturbation significantly modifies the properties of the complex network being perturbed . in the upper plateau region the perturbation by network p actually dominates the properties of the perturbed network s and reorganizes it , just as wiener anticipated . the pcm embodied in the cross - correlation cube therefore subsumes wiener s rule as we reviewed in west et al . in addition we showed the application of pcm to the phenomenon of habituation and other activities involving the human brain . aquino et al . ( 2011 ) observe that the growing interest in the dynamics of complex networks is shifting research attention from the synchronization of two stochastic units ( pecora and carroll , 1990 ) to the synchronization of large numbers of units ( wang , 2002 ) , an interesting phenomenon that is closely related to the very popular model of kuramoto ( 1984 ) . the single units of the processes of chaos synchronization are chaotic and they surprisingly synchronize while maintaining the erratic dynamics that they have in isolation . although the single units of the kuramoto model are regular , it is becoming increasingly evident that the emergence of a global synchronization is a condition independent of whether the single units are regular or stochastic . ( 2011 ) are poisson processes and if one of them drives the other , they would obey the principle of aperiodic stochastic resonance ( lukovic et al . , 2008 ) . if the two units are bi - directionally coupled they are expected to undergo a condition of perfect synchronization if the coupling is sufficiently intense . when the number of interacting units is very large a phase transition occurs from the non - cooperative to the cooperative behavior ( bianco et al . , 2008 ; turalska et al . , 2011 ) . it is important to stress that at criticality no permanent consensus is reached , and the mean value of the global field vanishes . yet ( no ) state for an extended time before making a transition to the no ( yes ) state . it is surprising that the phase transition literature seems to have overlooked , with only a few exceptions ( contoyiannis and diakonos , 2000 ; bianco et al . , 2008 ; turalska et al . , 2011 ) , that the transitions from the yes in other words , the time interval between two consecutive transitions is derived from a pdf that has the asymptotic time structure of eq . < < 3 . some authors ( bianco et al . , 2008 ; turalska et al . , 2011 ) argue that = 1.5 and others ( frantsuzov et al . , 2009 ) , releasing the condition that all the units share the same poisson rate , generate a global condition with crucial events characterized by < 2 , but significantly departing from the value = 1.5 . ( 2009 ) , might yield the misleading impression that the crucial value of is a consequence of ordinary statistical physics . ( 2011 ) is the discovery of a promising road to settle the problem of information transmission from one to another complex network . in fact , if the inner synchronization corresponds to a criticality condition and criticality generates crucial events with a power < 3 , then a complex network at criticality is a generator of 1/f noise , with a power spectrum s(f ) 1/f . thus , the problem of information transmission from one to another complex network becomes equivalent to the phenomenon of 1/f resonance illustrated in aquino et al . the experiments ( onsager , 1944 ; silvestri et al . , 2009 ) support the dynamical rather than the phenomenological lrt . it is important to stress that phenomenological lrt is a natural consequence of adopting the asymptotic time perspective replacing the waiting - times pdf ( ) of eq . 1 with ( ) 1/. this way of proceeding , although generating the elegant mathematics of fractional derivatives , has as an ultimate effect the misleading discovery of the death of linear response . this is not a unique way of connecting the longtime to the short - time regime . however , whatever form we adopt we are convinced that there will be a parameter corresponding to the parameter t of eq . it is reasonable to assume that an external perturbation may perturb either t or , or both . however , the perturbation of is incompatible with the assumption of a weak stimulus . in fact , is a consequence of the cooperation among the units of the network , and a perturbation may affect only if its strength is large enough to influence the interaction among the units of the network . thus , an external weak perturbation can only have an effect on t , thereby making the dynamical lrt become the proper way to study the response of a complex network to a weak external stimulus , in accordance with the experimental results ( onsager , 1944 ; allegrini et al . , 2009 ; silvestri et al . , 2009 ) . for these reasons , we can conclude that figure 1 is an original , and important , result of this research program . we hope that the application of the pcm may open the door to solving the problem of information transmission from one complex network to another , a research topic that is still in its infancy . in order to better understand the transfer of information between complex networks last year we numerically analyzing networks consisting of a large number of non - linearly interacting nodes . ( 2009 ) using a master equation formalism ( turalska et al . , 2011 ) were discussed where each element of the network is a two state oscillator and is described by a master equation of the form and pj(t ) is the probability of being in the state j = 1 ; p1 + p2 = 1 . the coupling functions are time - independent for non - interacting members of the network . subsequently , we considered a network consisting of l discrete variables located at the nodes of a 2d square lattice . each unit si is a stochastic oscillator and can be found in either of the above two states . for the dynamic complex lattice each element si interacts with each of its nearest neighbors and is updated in an elementary time step with transition rate g : here m is the total number of nearest neighbors ; m + 1 and m 1 are the number nearest neighbors that have made the decision yes and no , respectively . the single individual changes opinion , and as a consequence these numbers are variables fluctuating in time , while the total number of elements is constant . ( no ) makes a transition to the state no ( yes ) faster or slower according to whether the majority of the elements are in the state no ( yes ) or yes ( no ) , respectively . 2011 ) do all calculations on a l l lattice with periodic boundary conditions . they characterize the network in terms of the global order parameter whose variability is not dichotomous . in figure 2 an example of the temporal evolution for a single unit is compared with that of the global order parameter . ( a ) temporal evolution of a single unit and ( b ) of the global order parameter for the dmm realized on a square lattice with l = 50 , g0 = 0.01 , and k = 1.70 . ) note that the amplitude of the global order parameter depends on the value of the coupling constant k. when k = 0 , single units of the network are independent . when k > 0 , single units are less and less independent , resulting in a non - zero average . the quantity kc is the critical value of the control parameter k , at which point a phase transition to a global majority state occurs . in numerical calculations they use the time average eq |(t)| as a measure of the global majority . more precisely after an initial million time steps , which is sufficient time to suppress any transients , an average is taken over the same number of consecutive time steps in the dmm . they find that in the special case when m is the same for all the nodes and g0 1 , that dmm generates the same kind of phase transition as is observed in the 2d ising model discussed in onsager s seminal paper ( onsager , 1944 ) . the phase transition for the global variable eq is indicated in figure 3 under various conditions . it is evident that the dmm phase transition on a lattice is only equivalent to the ising model under very restricted conditions . the apparent equivalence between the ising and dm models is merely formal , since the dmm does not have a hamiltonian origin and its elements are not in contact with a thermal bath ( turalska et al . , 2011 ) . these differences explain why the equivalence requires the transition rate to vanish , so as to freeze the dynamics of the single units , in the absence of cooperation . the phase diagram for the global variable eq . the thin solid line and dashed line are theoretical predictions for the fully connected and 2d regular networks , respectively . in both cases l = and the latter case is the onsager prediction for a 2d regular lattice . the thick solid line corresponds to the global states observed for a 2d regular lattice with l = 100 and g0 = 0.01 . when the transition rate assumes a finite value the equivalence between dmm and the ising model is lost . ( 2011 ) investigate the parameter phase space to determine the domain of phase transitions and find that they can occur for values of k below that of the theoretical kc . there is also a situation for relative high transition rates in which every unit is surrounded by nearest neighbors in the opposite state , yielding an update of its state at every time step and generating the condition in which the order parameter is exactly zero at all times . ( 2011 ) conjecture that the crossings of the origin by the global order parameter are the significant events to observe . as illustrated in figure 2 they interpret the time interval between two consecutive crossings as the time duration of a given decision , even if this decision may rest on a slight and fluctuating majority . they evaluate the distribution density of decision time duration , ( ) , and the corresponding survival probability ( ) , where although emerging from a simple regular lattice , that is , one with no structural complexity , the survival probability presented in figure 4 shows a scale - free property that extends over more than four orders of magnitude in time for k kc a further increase in the coupling strength does not affect the power law region . survival probability function ( ) for the global order parameter evaluated on a 2d lattice with l = 50 , g0 = 0.01 and increasing values of the coupling constant k. the straight line corresponds to a slope of 0.50 , namely = 1.5 since ()1/. ( from turalska et al . wiener s rule maintains that a network with high information can organize one with low information . for example a tightly coupled organization , with rules and policies to cover all contingencies , changes little over time and therefore is low in information . cpm quantifies wiener s rule by introducing a measure of complexity allowing us to compare the level of information in interacting complex networks . law index of the hyperbolic distribution and a generalization of lrt enabled us to construct the cross - correlation cube to determine the degree of asymptotic influence one network has on another . in this way the 1/f variability of stimuli is found to resonate with the human brain ( grigolini et al . , 2009 ) , as when we are entranced by music or irritated by a dripping faucet ( grigolini and west , 2011 ; west and grigolini , 2011 ) . 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 . retinoblastoma ( rb ) is a primary intraocular tumor that is most commonly malignant in childhood ; this condition arises from primitive neural retinal cells destined to become photoreceptors and corresponds to 11% of all cancers in the first year of life . the incidence of rb worldwide is estimated at between 5,000 and 8,000 new cases per year , and although rb can occur at any age , this tumor occurs most often in preschool children ( 95% of cases diagnosed before 5 years of age ) [ 3 - 5 ] . the primary genetic event associated with rb is the inactivation of both alleles of the rb1 gene ( gene i d : 5925 , omim 614041 ) in the 13q14 locus by translocations , deletions , insertions , and point mutations . genetically , rb presents as hereditary ( 40% ) or non - hereditary ( 60% ) . in the inherited form , a constitutional mutated allele is always transmitted via germs ; meanwhile , in the non - hereditary form , both alleles are inactivated by somatic mutations . the tp53 gene ( gene i d : 7157 , omim 191170 ) is located at 17p13.1 and provides instructions for making the p53 tumor protein ( p53 ) ; this protein acts as a tumor suppressor and binds directly to dna . when p53 is damaged , other genes are activated to repair the damage . if the dna can not be repaired , this protein prevents the cell from dividing and sends signals for apoptosis ; thus , the protein has been nicknamed the guardian of the genome . genetic polymorphisms are variants of genome mutations that appear in certain individuals . these polymorphisms are transmitted to offspring and acquire a certain frequency in the population after many generations . the most common polymorphisms are single base changes , known as single nucleotide polymorphisms ( snps ) . the aim of this study was to determine the frequency and association between the snp rs9568036 ( the rb1 gene ) and the snp rs1042522 ( tp53 ) and clinical characteristics in a group of mexican children with rb . a total of 11 pediatric patients under 10 years of age with a diagnosis of rb confirmed by histopathological studies who presented at our hospital between march 2015 and august 2015 for close follow - up exams after completion of chemotherapy were recruited for polymorphism detection ( men 63.6% , mean diagnosis age=16 months , range 3 - 37 ; woman 36.4% , mean diagnosis age=20 months , range 4 - 42 ) . the mean duration of follow - up after diagnosis was 30 months ( range 4 - 87 ) . we excluded patients who had received blood transfusions in the previous 6 months or those who had received an organ donation . the study was approval by the local research ethics committee of high specialty medicine unit , specialty hospital ( umae ) no . 71 of mexican institute of social security ( imss ) with code registered r201550170 20 , and informed consent was obtained from the responsible caregiver for all cases . the study was conducted in accordance with the declaration of helsinki and in compliance with the laws and regulations of the mexican general law of health in research for health . fifteen microliters of venous blood of patients with rb were obtained , placed in 100 l of cell lysis solution , and immediately stored at 2 c . polymorphism analysis was performed by amplification of the polymorphic site with conventional pcr using gotaq master mix ( promega inc . , madison , wi ) to obtain similar amounts of amplification products from each sample . the 15 l reaction contained 7.5 l gotaq master mix , 5.7 l water , 0.3 l the each primer ( aag gga ggg ata gca tta gga ( sense ) , gaa tgt gca ggt ttg tta cat agg ( antisense ) for rb1(rs9568036 ) and cat cac acc ctc agc atc tc ( sense ) , gcc tgg tca gga gct tat tt ( antisense ) for tp53 ( rs1042522 ) ) and 1.2 l blood lysate . high - resolution melt ( hrm ) analysis , which is based on the characterization of the pcr products according the dissociation behavior of the dna strand , was performed because this method is sensitive even to a simple change of base . a melting temperature ( tm ) of 55 c to 95 c the conditions of the conventional pcr amplification were : initial denaturation at 94 c for 10 min , followed by an amplification of 55 cycles of 95 c / 45 s ; 54 c / 45 s ; 72 c / 45 s. the reaction mixture for each sample ( 15 l ) was prepared according to the manual accompanying the hrm type - it reagent ( qiagen inc . , germantown , md ) , and contained 7.5 l , 5.9 l water , 0.3 l sense primer , 0.3 l antisense primer , and 1 l pcr product ( table 1 ) . the conditions of a second amplification using real - time pcr were the same that conventional pcr , only that they used 20 cycles for both polymorphisms ; for the analysis of the melt curves a rotorgene thermocycler ( quiagen inc . dna melting curve analysis between 55 c and 90 c for both genes was used as an internal control to prevent false - negative or false - positive results ( figure 1 ) . as the temperature increased , the strands of the amplicon separate to form single - stranded dna , causing the fluorescent intercalating dye to dissociate from the dna and stop fluorescing . the snps rs9568036 ( rb1 ) and rs1042522 ( tp53 ) selected for the study were taken from the hapmap snp database and were genotyped by iselect hd bead - chip ( illumina , san diego , ca ) . statistical analysis included measures of central tendency and dispersion ( p values less than 0.05 were considered significant ) , and the chi - square and student t tests were used to determine the association between variables using spss version 19.0 software ( spss inc . , genotype and allele frequencies , as well as hardy weinberg equilibrium , were evaluated using guo and thompson s method . the frequencies of genotypes and alleles obtained for each of the examined snps are presented in table 2 . there was a statistically significant difference ( p<0.05 ) between the frequency of the genotypes and the expected genotypes in the population according to the hardy weinberg equilibrium ( table 3 ) . there was a statistically significant difference between the rb1-gg / rs9568036 polymorphism and tumor chemoresistance , as well as between the rb1-aa and ag / rs9568036 polymorphisms and tumor chemosensitivity ( table 4 ) . there was no statistically significant association ( p = 0.6576 ) between the rb1/rs9568036 polymorphism and any allelic variants with unilateral or bilateral disease presentation . there was a statistically significant relation between unilateral presentation of the disease and the polymorphism tp53-cc / rs1042522 ( p = 0.0386 ) , rate ratio ( rr ) = 2.0 ( 95% ci = 0.755.32 ) , but the allelic variants tp53-cg and gg / rs1042522 had no relation with the laterality of the disease ( p = 0.4611 ) . / rs9568036 and men ( p = 0.0386 ) , rr = 2.0 ( 95% ci = 0.765.32 ) , as well as between the rb1-aa and ag / rs9568036 polymorphisms and women ( p = 0.0027 ) , rr = 8.0 ( 95% ci = 1.2850.04 ) . the rb1 gene plays an important role in the negative control of cell cycling and tumor progression by translating the rb protein and inhibiting cell division . in the hypophosphorylated state , the rb protein is active and prevents the attachment of an e2 factor ( e2f ) transcription factor ( related to dna replication for division ) . being hyperphosphorylated , the rb protein receives kinases phosphate groups and is inactivated , allowing cells to move from the g1 to the s phase and proceed with division . the absence of rb protein activity , as a result of mutations that inactivate or deregulate its phosphorylated state , causes continuous cell division and leads to a large number of human cancers and metastasis , including cellular response to mitotic inhibition induced cell death during chemotherapy . to have equal concentrations of dna , the first amplification was performed with end - point pcr , after which the pcr - hrm technique was performed to analyze the polymorphisms . the pcr - hrm technique used to analyze the polymorphisms in this study has been used in different clinical applications and successful investigations even in the analysis of snps in different types of cancer . each double - stranded dna fragment presents a characteristic fusion pattern , which is determined by the content , length , and position of the cgs in the dna sequence , thus allowing the detection of minimal alterations in the sequences , making this method faster , simpler , and less expensive compared with other methods used for amplification and quantification of dna sequences . liu and colleagues studied some polymorphisms of the rb1 gene and found that in response to platinum- and taxane - based chemotherapy , patients with the polymorphism rs4151510 gg had better survival rates for squamous cell lung cancer , while patients with the polymorphisms rs4151510 , rs4151465 , and rs9568036 had increased survival rates for non - small lung cancer cells . in this study , we found that the polymorphism rb1/rs9568036 homozygous mutant gg was associated with twice the likelihood of rb in men refractory to conventional chemotherapy , while the snp rs9568036 homozygous wild aa and heterozygous ag were associated with tumor sensitivity to chemotherapy . found that in rb the polymorphisms rs1801270 c > a and rs1059234 c > t of the cdkn1a ( gene i d : 1026 , omim 116899 ) gene were associated with an increased risk of rb ( odds ratio [ or ] = 2.5 , 95% ci = 1.384.53 ) , whereas patients with the cc genotype for both polymorphisms had a lower risk of rb ( or = 0.43 , 95% ci = 0.250.74 ) . in this study , we found that the polymorphism rb1/rs9568036 homozygous wild aa and heterozygous ag were associated with an increased likelihood ( 8x ) of rb in women . kadam - pai and colleagues studied the tsp509i polymorphism of the rb1 gene in eight asian populations and found an ethnic variation prevalent in southeast asia that confers increased susceptibility to rb . in the present study of a northern mexican population , we found that the rb1/rs9568036 polymorphism and the allelic variants rb1-gg / rs9568036 , rb1-aa , and ag / rs9568036 were associated with an increased risk for rb . furthermore , most of the somatic mutations in the tp53 gene alter the individual amino acids in p53 leading to the production of an altered version of the protein that can not bind dna efficiently . this defective protein can accumulate in the nucleus of cells and prevent them from suffering apoptosis in response to dna damage . thus , the damaged cells continue to grow and divide an unregulated manner . in the rs1042522 polymorphism of the tp53 gene , the substitution g > c exists in codon 72 and exon 4 . han and colleagues found that the gg genotype of the snp rs1042522 of the tp53 gene showed increased resistance to first - line chemotherapy compared with those with the genotypes cg or cc ( 60% versus 27% , p = 0.014 ) in a study of 148 patients with lung cancer with non - small cell carcinomas . shabazz and colleagues found a correlation between an increased risk of breast cancer and allelic variants of the rs1042522 polymorphism in women from bangladesh ( p = 0.0053 , or = 1.69 ) . meanwhile , chen et al . reported in a chinese population that this polymorphism is associated with local tumor invasion . in this study , we found a statistically significant relation between the unilateral presentation of the disease and the same snp . the rs9568036 polymorphism in the rb1 gene and its allelic variants can be associated with the type of response to medical therapy and associated with male sex , while the allelic variant rs1042522 polymorphism in the tp53 gene is associated with the unilateral presentation of the disease in a group of mexican children with rb . the rs9568036 polymorphism in the rb1 gene and its allelic variants can be associated with the type of response to medical therapy and associated with male sex , while the allelic variant rs1042522 polymorphism in the tp53 gene is associated with the unilateral presentation of the disease in a group of mexican children with rb .

the army research laboratory program on the network science of human decision making brings together researchers from a variety of disciplines to work on a complex research problem that defies confinement within any single discipline . consequently , new and rewarding solutions have been obtained for a problem of importance to society and the army , that being , the human dimension of complex networks . this program investigates the basic research foundation of a science of networks supporting the linkage between the cognitive and social domains as they relate to human decision making . the research strategy extends recent methods of non - equilibrium statistical physics to non - stationary , renewal stochastic processes characteristic of the interactions among nodes in complex networks . the theoretical analyses of complex networks , although mathematically rigorous , often elude analytic solutions and require simulation and computation to analyze the underlying dynamic process . the information transfer between two complex networks is calculated using the principle of complexity management as well as direct numerical calculation of the decision making model developed within the project . purposeto determine the frequency and association of polymorphisms in the tp53 and rb1 genes with clinical characteristics in a group of children with retinoblastoma ( rb ) in northern mexico.methodsa prospective , longitudinal , and analytical study of 11 patients diagnosed with rb was conducted . endpoint pcr and high - resolution real - time pcr were performed . chi - square and student t tests were used to evaluate associations between variables . allelic frequencies , as well as genotypic and hardy weinberg equilibriums , were evaluated using guo and thompson s method.resultswe found a statistically significant difference between the polymorphism rb1-gg / rs9568036 and tumor chemoresistance ( p<0.05 ) . the allelic variants rb1-aa and ag / rs9568036 were determined to be associated with tumor chemosensitivity ( p<0.05 ) . a statistically significant relation between the polymorphism rb1-gg / rs9568036 and males ( p = 0.0386 ) , rate ratio ( rr ) = 2.0 ( 95% confidence interval [ ci ] = 0.765.32 ) , as well as between the allelic variants rb1-aa and ag / rs9568036 and females ( p = 0.0027 ) , rr = 8.0 ( 95% ci = 1.2850.04 ) , was observed . we also observed a statistically significant association between the rs1042522 polymorphism in the tp53 gene and unilateral presentation of the disease.conclusionsthe rs9568036 polymorphism in the rb1 gene and the allelic variants can be associated with type of response to medical therapy and associated with male sex , while the allelic variant rs1042522 polymorphism in the tp53 gene is associated with the unilateral presentation of the disease in a group of mexican children with rb .

the nose is a central structure of the face , so attempts to reconstruct ideal form and esthetics can be complicated . the majority of patients with cleft lip nose deformity require augmentation rhinoplasty for a more balanced nasal dorsum . traditionally , autogenous cartilages from various sites are used to correct cleft lip nose deformity . however , sometimes results of corrective rhinoplasty using auricular or septal cartilages are unsatisfactory due to the weakness of the graft material . in that case , rib cartilage could be a good material of choice for corrective rhinoplasty for cleft lip nasal deformity . however , the harvesting procedure leaves a cutaneous scar that is unacceptable , especially for a young female patient . so we need to find alternative surgical options to replace the autogenous rib cartilage . in 1998 , a new biomaterial called permacol ( tissue science laboratories plc , aldershot , uk ) was licensed in europe for permanent implantation in human body . permacol consists of acellular , cross - linked porcine dermal collagen that is resistant to degradation by collagenase . it had been widely used in general and gynecological surgery in the treatment of hernia and otolaryngology surgery such as surgical closure of nasal septal perforation . recently it was described in facial contour augmentation surgery including esthetic augmentation rhinoplasty and reconstruction of a post - traumatic nasal deformity . the author augmented nasal dorsum after reconstruction of the lower third of the nose during corrective rhinoplasty in patients with cleft lip nasal deformity . in this report we describe three cases of rhinoplasty procedures using autogenous auricular cartilage and synthetic porcine dermal collagen with highlights of how to use it . a 17-year - old female patient with secondary deformity due to unilateral incomplete cleft lip and palate was referred to the department of oral and maxillofacial surgery , gangneung - wonju national university dental hospital . five years ago , she underwent autogenous bone graft on her cleft alveolus . during this time , her chief complaint was the deformed nose , so we decided upon corrective rhinoplasty and minor revision surgery on her upper vermilion . after taking standard rhinoplasty photograms , formal consent for the use of xenograft material was obtained . the operation was performed under general anesthesia with additional anesthetics consisting of 2% lidocaine and 1:100,000 epinephrine in the planned dissection area . via an open rhinoplasty approach with intercartilaginous and stair - shaped transcolumellar incision ( fig . 1a ) , the lower lateral cartilage was dissected from surrounding soft tissues and prepared a subperiosteal pocket for the xenograft on nasal bone ( fig . the permacol sheet comes ready to use and can be cut to the desired size easily . we inserted two layers ( 3 mm ) of permacol under the dorsal skin of the nose , and fixed it to the underlying cartilages with 4 - 0 polydioxanone ( fig . after closing the surgical wounds , we applied external nasal stent and tape for two weeks , as well as a nasal conformer for six months at night postoperatively . esthetic results in visits at one month , six months , and one year were satisfactory for patients and surgeon ( fig . 2 ) . the dorsal implant was intact and did not show any resorption or displacement . a 17-year - old male patient with secondary deformity due to unilateral he had undergone several surgeries including distraction osteogenesis for collapsed maxilla , secondary alveolar bone grafting for cleft alveolus , and pharyngoplasty by the author . on this occasion ( january 25 , 2009 ) , corrective rhinoplasty was performed . under general anesthesia , open rhinoplasty was performed via an intercartilagenous incision , continuous with the transcolumellar incision on the line of previous scar ( fig . 3c ) , we applied it as a columellar strut and gull - wing type graft ( fig . 4 ) . a 14-year - old male patient with secondary deformity due to bilateral cleft of primary palate presented to the department . his mother s chief complaint was the visible scar on upper vermilion and the compressed nose , so we decided to perform a cheilorhinoplasty . under general anesthesia , open rhinoplasty was done via an intercartilagenous incision inside both nostrils and the incision for scar revision ( fig . , we applied it as a columellar strut and gull - wing type graft for prominent nasal dome ( fig . we then implanted two layers of permacol in the form of an onlay graft , and fixed it to the grafted auricular cartilages with monocryl sutures ( fig . a 17-year - old female patient with secondary deformity due to unilateral incomplete cleft lip and palate was referred to the department of oral and maxillofacial surgery , gangneung - wonju national university dental hospital . five years ago , she underwent autogenous bone graft on her cleft alveolus . during this time , her chief complaint was the deformed nose , so we decided upon corrective rhinoplasty and minor revision surgery on her upper vermilion . after taking standard rhinoplasty photograms , formal consent for the use of xenograft material was obtained . the operation was performed under general anesthesia with additional anesthetics consisting of 2% lidocaine and 1:100,000 epinephrine in the planned dissection area . via an open rhinoplasty approach with intercartilaginous and stair - shaped transcolumellar incision ( fig . 1a ) , the lower lateral cartilage was dissected from surrounding soft tissues and prepared a subperiosteal pocket for the xenograft on nasal bone ( fig . the permacol sheet comes ready to use and can be cut to the desired size easily . we inserted two layers ( 3 mm ) of permacol under the dorsal skin of the nose , and fixed it to the underlying cartilages with 4 - 0 polydioxanone ( fig . after closing the surgical wounds , we applied external nasal stent and tape for two weeks , as well as a nasal conformer for six months at night postoperatively . esthetic results in visits at one month , six months , and one year were satisfactory for patients and surgeon ( fig . 2 ) . the dorsal implant was intact and did not show any resorption or displacement . a 17-year - old male patient with secondary deformity due to unilateral complete cleft lip and palate was referred to our department . he had undergone several surgeries including distraction osteogenesis for collapsed maxilla , secondary alveolar bone grafting for cleft alveolus , and pharyngoplasty by the author . on this occasion ( january 25 , 2009 ) , corrective rhinoplasty was performed . under general anesthesia , open rhinoplasty was performed via an intercartilagenous incision , continuous with the transcolumellar incision on the line of previous scar ( fig . 3c ) , we applied it as a columellar strut and gull - wing type graft ( fig . a 14-year - old male patient with secondary deformity due to bilateral cleft of primary palate presented to the department . his mother s chief complaint was the visible scar on upper vermilion and the compressed nose , so we decided to perform a cheilorhinoplasty . under general anesthesia , open rhinoplasty was done via an intercartilagenous incision inside both nostrils and the incision for scar revision ( fig . , we applied it as a columellar strut and gull - wing type graft for prominent nasal dome ( fig . we then implanted two layers of permacol in the form of an onlay graft , and fixed it to the grafted auricular cartilages with monocryl sutures ( fig . there is a vast number of surgical techniques and grafting materials for cleft lip nose deformity reconstruction . in general , autogenous tissues remain the gold standard for reconstructive surgery of cleft lip nose deformity . especially for augmentation of the nasal dorsum however , autogenous grafting procedures require additional surgery and donor site morbidity , and sometimes graft material is limited . thus maxillofacial plastic and reconstructive surgeons always need to prepare allogeneic or alloplastic materials as an alternative . silicone , high - density porous polyethylene , expanded polytetrafluoroethylene , and human dermal collagen are used as alloplastic materials for augmentation rhinoplasty , and have both advantages and disadvantages . permacol can be a useful material for augmentation rhinoplasty because it is readily available in the required volume , it is easy to shape , and it seems to maintain the structural integrity of the nose . in this study , three patients underwent corrective rhinoplasty via external approach using autogenous auricular cartilage and permacol composite grafting . the original dimension of the permacol sheet used was 60301.5 mm , and two layers of permacol were inserted into the nasal dome with customized size for each patient . if layering is required , the material can be cut into separate sheets , creating edges rather than folding or rolling , probably due to possible colonization of microorganisms . , the auricular cartilage was covered with a double layer of permacol to form the ideal nasal contour . afterwards , the author felt two layers of permacol was aesthetically sufficient for the patient with thin nasal skin ( case 1 , 3 ) , but deficient for the patient with thick facial soft tissues ( case 2 ) . previous studies demonstrate that permacol , when implanted in human body , is readily surrounded by host tissues such as fibroblasts and vascular endothelial cells . hunter et al . observed a permacol explant 15 months post implantation , originally inserted for augmentation rhinoplasty . macroscopically , permacol was covered with a clear layer of fibrous tissue , characterized histologically by the irregular arrangement of collagen as well as the presence of some cells and elastin fibers . suggested that permacol acted as a matrix into which autogenous tissue could grow . from those studies and our own limited experiences with three patients , inserted fourty - five permacol implants for augmentation rhinoplasty over a 4-year period , with only one case of postoperative implant removal . they concluded that permacol seemed to be a biocompatible alternative to conventional grafting materials , with minimal morbidity . compared to the removal rate of silicone and the infection rate of gore - tex , pitkin s clinical results are encouraging . moreover , permacol can be safely used in a contaminated field . in our study , immediately after the operation , no patients complained of any foreign body sensations or discomfort at usual daily activities . . recommended long - term follow - up for the use of permacol because some graft absorption was noted in their patient , who had undergone nasal profile augmentation . the author followed three patients for more than five years , but there was no sign of graft absorption . hopkins et al . also report successful long - term use of permacol in rhinoplasty . in conclusion , we obtained pleasing results in reconstruction of cleft lip nose deformity with simple and safe surgical procedures . in our limited experience with three patients , the author concludes that the permacol can be a reliable surgical option for patients with cleft lip nose deformity . prostate cancer ( pca ) is a frequent cancer that can be cured if early diagnosed ( 1 ) . prostate specific antigen ( psa ) has become an important tool in pca screening ( 2 , 3 ) and men with serum psa greater than 4ng / ml are at higher risk of pca . these patients are usually referred for a prostate biopsy ( bxp ) . however , increased psa levels are also associated with conditions other than cancer ( 3 ) , such as benign prostatic hyperplasia ( bph ) and prostatitis ( 4 , 5 ) . chronic abacterial prostatitis is a common diagnosis in men of all ages , with widespread demographics , and it is a common reason for yearly visits to the doctor in the united states ( 3 ) . only a few studies have linked prostatitis to an increase in serum psa ( 6 - 9 ) . subclinical inflammation of the prostate could elevate serum psa in asymptomatic patients , confounding the use of psa values to indicate bxp ( 10 ) . in the majority of cases , prostatitis is an incidental pathological finding that has no clinical relevance . there has been investigation into ways to decrease the misleading diagnosis resulting from inflammation . repeat psa measurements after a period of observation in asymptomatic men can help to avoid unnecessary bxp ( 10 - 12 ) . it has been suggested that antibiotic therapy ( at ) can also avoid bxp in many patients with prostate inflammation , in the psa grey zone ( 4.0 to 10.0ng / ml ) ( 6 , 13 , 14 ) . currently , the indications of re - bxp in patients with a negative initial biopsy are few ; therefore , the first bxp must be precise . in an effort to improve the reliability of psa reduction as an indicator , and consequently avoid unnecessary prostate biopsy , we conducted a prospective , controlled , non - randomized study to evaluate the effect of at on psa levels in patients who have an initially mild psa elevation ( 4.0 - 10ng / ml ) . a prospective , controlled , non - randomized trial was carried out , with 106 asymptomatic men with total psa ( tpsa ) levels between 4.0 and 10.0ng / ml , who underwent routine evaluation from april 2007 to october 2011 . the criteria for inclusion in the trial were digital rectal examination ( dre ) with no suspicion of malignancy and no history of urologic instrumentation , use of antibiotics , and urinary infection or sexually transmitted disease in the previous 12 months . patients with diseases like diabetes or alcoholism , those describing the use of illegal drugs , those undergoing treatment for bladder outlet obstruction and patients with previous bxp ( positive and negative ) were excluded , as were those participants who did not use the at correctly or who did not carry out the follow - up correctly . after institutional review board ( ethical committee ) approval , all study participants provided informed written consent before enrolment . after the initial consultation , the serum levels of total psa ( tpsa ) and free psa ( fpsa ) were determined . the body mass index ( bmi ) , free psa fraction ( % fpsa ) , and psa density ( dpsa ) the participants were divided into two groups ( figure-1 ) according to their decision regarding the use of antibiotics . this decision was made after the authors explained about the lack of evidence for this treatment . group i patients received antibiotics , while group ii patients chose not to use at . in group i , 64 men used ciprofloxacin 500 mg twice a day for a period of three weeks . then , three weeks after the end of at , the serum psa , dpsa , and fpsa were again determined ( figure-1 ) . the results of these patients were compared with those of 42 participants with psa between 4.0 and 10.0ng / ml whose exams were repeated after 6 weeks , without antibiotics , at the participant s discretion . psa was considered to be normalized after treatment when the values returned to lower than 4ng / dl . all of the participants underwent bxp 2 - 4 weeks after the second psa determination . based on the pre- ( psapre ) and post - treatment ( psapost ) psa values , the variation ( psavar ) thus , the variation rate ( psavar ) can be calculated with psa = ( psavar / psapre).100% . the bxp was ultrasound - guided and a minimum of 12 cores were sampled with determination of prostate volume in cubic centimetres ( cc ) . for those participants whose biopsy did not show pca , dre : digital rectal examination ; tpsa : psa total ; fpsa : psa free . the data obtained were analysed by testing the difference between two proportions for incidence of pca in each group , and evaluation of the decrease in psa levels . the other data were evaluated using the student t test , citing the critical t for a significance level of 0.05 . the data obtained were analysed by testing the difference between two proportions for incidence of pca in each group , and evaluation of the decrease in psa levels . the other data were evaluated using the student t test , citing the critical t for a significance level of 0.05 . the age of the participants ranged from 47 to 78 years , with a mean age of 66.1 years and median age of 61.8 years . the mean age of g1 was 61.8 years and of g2 was 62.6 years ( p=0.60 ) . the prostate volume was 51.123.8 gr in g1 and 53.919.2 gr in g2 ( p=0.52 ) . the diagnosis of pca was confirmed in 25 of the 106 participants ( 23.6% ) ; in 9 out of 42 participants in g2 ( 21.4% ) and in 16 out of 64 in g1 ( 25% ) ( z=0.42 ; p>0.05 ) . group igroup iipwith atb meansdwithout atb meansdage ( years)61.817.8362.576.370.6bmi ( kg / m)27.863.5129.668.090.02volume ( cc)51.1523.8753.9719.280.527psapre6.821.666.761.680.429psapost5.291.85.382.160.409fpsapre1.250.471.310.400.233fpsapost1.030.441.060.480.383dpsapre0.150.050.140.060.261dpsapost0.110.060.110.070.369values expressed in mean standard variation . student t test there was a more than 10% psa decrease in 72 out of 106 patients ( 67.9% ) ; this rate was 65.6% and 71% in groups 1 and 2 , respectively ( z=-0.63 ; p>0.05 ) . of the 42 participants from g1 in which the psa decreased after at , 6 ( 14.3% ) had a positive and 36 ( 85.7% ) a negative biopsy . in g2 , 30 participants had a psa reduction , with 4 ( 13.3% ) with a positive and 26 ( 86.7% ) a negative biopsy . after the use of at , the psa level decreased to < 4ng / ml in 26 participants ( 24.5% ) ; in 15 ( 23.4% ) of those who used at , and in 11 ( 26% ) from g2 ( z=-0.032 ; p>0.05 ) . the percentage of participants who had a value below 2.5ng / ml in the second psa was 9.4% for g1 , and 7.1% for g2 ( z=0.40 ; p>0.05 ) . the initial and final values for psa , fpsa , % fpsa and dpsa decreased significantly in both groups ( table-2 ) . table 2comparison of initial and final psa values between groups.group igroup ii pre - atbpost - atbppsa initialpsa finalptpsa6.821.665.291.8<0.0016.761.685.382.16<0.0001fpsa1.250.471.030.440.00021.310.41.060.480.0003%fpsa18.265.0820.136.350.001719.915.5621.086.950.072dpsa0.150.050.110.06<0.00010.140.040.110.070.0003values expressed in meanstandard variation . student t test the mean total psa in g1 was 6.821.66 and 5.291.8ng / ml before and after treatment , respectively ( p<0.001 ) ; this represents a psavar of -1.531.93ng / ml . in g2 , the mean psa before and after antibiotics was 6.761.68 and 5.382.16ng / ml , respectively , with a psavar of -1.381.87 ( p<0.001 ) . the psa in g1 was 25.56 , and in g2 was 28.7 ( p=0.429 ) . the differences between g1 and g2 are shown in table-2 , according to the initial and final fpsa and dpsa . there was no statistical difference between these values . in relation to dpsa , for g1 , the initial mean was 0.150.05ng / ml / gr of prostate , which decreased to 0.110.06ng / ml / gr ( p<0.0001 ) . in g2 , these figures were 0.140.04ng / ml / gr and 0.110.07ng / ml / gr , respectively ( p=0.0003 ) . these same comparisons , analysing the cases with and without pca , are shown in table-3 . in the participants diagnosed with pca , there was no statistical difference in relation to variation in psa , fpsa , % fpsa or dpsa . however , in the participants with negative bxp , there was a significant reduction in psa , fpsa and dpsa , but not in % fpsa . table 3comparison of psa values between patients with and without prostate cancer.benignprostate cancer psa initialpsa finalppsa initialpsa finalptpsa6.851.724.961.980.0016.631.496.521.190.2fpsa1.350.441.060.470.0011.020.370.970.400.077%fpsa19.965.0522.205.890.12415.524.7515.015.690.641psad0.140.060.100.06<0.000010.150.020.150.070.61values expressed in meanstandard variation . student t test the sensitivity to a decrease greater than 10% in psa after the use of at to a diagnosis of prostate cancer was 31% , with a specificity of 23% ; the positive predictive value ( ppv ) was 12% and the negative predictive value ( npv ) was 23% . regarding to the possibility of reducing unnecessary bxp after at , it should be emphasized that none of the 25 participants with pca had a final psa below 4ng / ml . the present study analysed the effect of at on psa ( tpsa , fpsa , % fpsa and dpsa ) and investigated whether a relevant psa reduction induced by at could be related to a decreased cancer detection rate at biopsy . physiological conditions other than cancer can cause an increase in serum psa levels that lead to potentially unnecessary biopsy procedures , increasing inconvenience for the patient , and causing over - diagnosis , over - treatment and elevated medical costs ( 15 ) . prostate cancer is determined in only 34% of biopsies performed on the basis of psa elevation ( 1 ) , and in 20 - 30% in patients with normal dre and psa values of between 4 and 10ng / ml . therefore , there is a high level of unnecessary biopsies , particularly in this group ( 16 , 17 ) . the literature demonstrates a relationship between acute and chronic inflammation with elevated psa , but there have been recent studies that suggest the effects and benefits of chronic prostatitis treatment on psa ( 18 - 21 ) . in our study , pca was diagnosed more frequently in patients treated with at ( 25% versus 21.4% , p>0.05 ) , but without statistical relevance . scardino ( 22 ) suggested that the changes in psa with at were similar to the random variations found in healthy men . also , potts ( 23 ) demonstrated no significant differences in bacterial cultures before or after at between psa responders and non - responders . okada ( 24 ) and schatteman ( 25 ) concluded that subclinical inflammation could cause psa elevation , and emphasized the fact that nearly half of all clinically asymptomatic men with elevated psa levels have laboratory signs of prostatitis . they suggest that the use of antibiotics would result in a decrease in psa levels in almost 50% of patients , thereby avoiding bxp . this approach , however , requires careful follow - up , especially for patients whose psa levels fail to decrease to within the normal range ( 26 , 27 ) . kaygisiz ( 1 ) and del rosso ( 19 ) suggested that at should be administered for 3 weeks , regardless of the presence of inflammation when psa levels are in the grey zone , before making a decision regarding whether or not to carry out a biopsy . on the other hand , serretta et al . ( 28 ) found no cancer present if psa levels decreased to below 4ng / ml , or more than 70% , and postulated that biopsy can be postponed , with only a small risk of failing to detect cancer . in multivariate analysis with other clinical variables , although this was not a randomized trial , it was prospective , assessing asymptomatic males without a clinical indication of prostatitis ; the study demonstrated that a large reduction in psa following antibiotics may help to avoid biopsy in selected patients in whom the psa elevation is probably due to inflammation / infection . our data show that those patients who received at and those who did not had the same rate of normalization of psa ( < 4ng / ml ) . , psa returned to normal levels in 23.4% of patients , compared with 26% patients in the non - treatment group . the reason for this is still unclear , although psa normalization does not rule out pca diagnosis , and biopsy must still be considered . magri et al . ( 29 ) , showed that the presence of bph may prevent the reduction of psa induced by combination pharmacological therapy , and suggest that care must be taken in the adoption of psa as a marker of therapeutic efficacy in the presence of confounding factors like bph . according to these authors , psa should be used as a significant component of a strategy that integrates multiple diagnostic approaches . ( 11 ) showed a psa reduction in 63% of patients following at , with psa returning to normal values in 9% of cases , thus avoiding prostate biopsy . ( 23 ) documented psa normalization in 42% of patients , and brett et al . these studies did not perform bxp in all patients to exclude the diagnosis of pca after treatment . however , our study performed biopsy in all patients ( treated and not - treated ) , comparing the pca incidence between groups . however , the entry of participants was not random , but based on the decision made by each participant ; this may represent a selection bias . moreover , the shared decision with the patient is one of the commonalities in international guidelines . approximately 10 - 15% of men will have a psa level > 4ng / ml in any given round of screening . however , the level will return to normal in the subsequent test in 26 - 37% , and will become normal with the next testing in 40 - 55% . ( 31 ) showed that psa levels tend to fall when repeated after 45 days , regardless of at . once normalized , 65 - 83% of men have normal psa levels for several years without therapy ( 32 ) . if psa levels do not fall , the probability of finding cancer is higher than if levels decrease . this occurs because pca is more likely to occur in men with sustained psa elevation than in those with a randomly variable psa that is temporarily elevated ( 24 ) . our study shows a lower , but not significant , cancer detection rate in patients with decreased psa , fpsa and dpsa after antibiotic therapy , demonstrating a correlation between psa normalization and prostatitis or negative biopsy . a psa reduction rate of 10% occurred in 58.5% of patients ; however , it was lower in patients who received antibiotics than in those who received no treatment ( 65.6% versus 71% ) , but there was not significant difference ( p>0.05 ) . the sensitivity , specificity and accuracy of a psa reduction rate of 10% were 31% , 23% and 25% , respectively . this level is therefore not recommended as a cut - off point for clinical decision - making . ( 28 ) showed a significantly lower cancer detection rate in patients with decreased psa after antibiotic therapy , demonstrating a correlation between psa reduction and negative biopsy , with an odds ratio varying from 1.2 to 3.9 for reduction percentages of between 10 and 90% . they suggested that a psa reduction rate of 50% can be adopted and 11% of biopsies avoided until a further psa increase occurs . ( 33 ) reported that the f / t psa ratio appears to be more suggestive of pca than psa in these cases . it should be emphasized , however , that a long follow - up time is needed to determine whether any of these men will have prostate cancer in the near future , and wider studies are required to identify the optimal psa reduction level at which biopsy can be postponed . ( 35 ) claimed that bph and prostatitis appear to be more frequent causes of psa elevation . scardino ( 22 ) recommended that asymptomatic men presenting with a modestly elevated psa level ( < 10ng / ml ) and a normal digital rectal examination could be reassured and then the psa level could be repeated once or twice ; if the levels remained elevated , this would be an indication of the need to perform a biopsy . ( 36 ) , in a prospective randomized and double - blind trial with placebo , demonstrated that psa reduction occurred after antibiotic and placebo application , and suggested that a decrease in psa does not indicate the absence of pca . recently , faydaci et al . ( 37 ) demonstrated that at given to patients with psa levels higher than the threshold value has not led to a significant change in prostate needle biopsy decisions , and suggested that bxp should be considered without the use of at in patients with high psa values if a suspicion of prostatitis does not exist . the literature does not support the evidence that antibiotics alter psa levels except in the presence of bacterial prostatitis , which is an uncommon condition . we should wait for a second psa assessment before prostate biopsy in asymptomatic male patients once the psa will spontaneously reduce in a quarter of cases ; and that antibiotic use has no role in this clinic scenario ( 36 ) . there are several implications in the use of empiric at for patients with elevated psa levels . scardino ( 22 ) emphasized some disadvantages of this approach , such as cost , toxicity , and the fact that it can cause complications of infection . moreover , a decrease in psa after at does not absolutely exclude the presence of pca , even if the psa decreases to very low levels . in addition , there is concern that the indiscriminate use of empiric antibiotics could lead to the development of resistant bacterial species and thereby expose the patient to more resistant and aggressive sepsis , should a biopsy eventually be performed ( 38 - 40 ) . empirical antibiotic therapy in asymptomatic male patients is not related to psa reduction and that psa reduction after antibiotic can not postpone prostate biopsy . based in our findings additionally , it is not possible to define a safe rate reduction and further studies stratifying the relative values of reduction and cancer risk are needed .

esthetic reconstruction of cleft lip nose deformity is a challenging task in surgical management of patients with orofacial cleft . the author reconstructed cleft lip nose deformity effectively using autogenous auricular cartilage and a relatively new graft material of porcine dermal collagen , permacol. after correction of the deformed lower third of the nose with patient s auricular cartilage , we applied permacol to augment the entire nasal dorsum . three patients were treated and followed for up to five years . all patients improved in nose aesthetics without any inflammatory or immunogenic reaction . the author suggests that the use of permacol for nasal profile augmentation in the treatment of cleft lip nose deformity is an alternative surgical strategy with minimal surgical invasiveness . the author report long - term experience with combined use of auricular cartilage and permacol in nasal reconstruction for cleft lip nose deformity . purpose we investigated the effect of antibiotics on psa in asymptomatic patients with mild psa elevation.materials and methods we prospectively evaluated , in a non - randomized design , 106 asymptomatic patients with psa of 4 - 10ng / ml , with a negative digital rectal examination and with no urinary tract infection evidence for 2 years . patients were divided into two groups : those treated with antibiotics for 3 weeks ( g1 ) and those who were not treated ( g2 ) . psa was taken six weeks after and prostate biopsy was performed in all patients.results pca was diagnosed in 25 of 106 patients ( 23.6% ) : 16 ( 25.0% ) in g1 and 9 ( 21.4% ) in g2 ( p>0.05 ) . psa normalization was experienced in 24.5% . in g1 , psa returned to < 4ng / ml in 15 ( 23.4% ) patients compared to 11 ( 26% ) patients in g2 . in the patients with a positive biopsy , no significant variation was noted in psa , fpsa , % fpsa and dpsa after antibiotic treatment . a significantly lower cancer detection rate was noted with decreased psa , fpsa , and dpsa after antibiotic use . a psa reduction rate of 10% occurred in 58.5% , and this was similar in both g1 and g2 groups . the sensibility , specificity and accuracy of psa reduction of 10% were 31% , 23% and 25% , respectively.conclusion empirical antibiotic therapy in asymptomatic male patients is not related to psa reduction . the greater than 10% psa reduction after antibiotic in this population can not postpone prostate biopsy .

chronic myeloproliferative neoplasms ( mpns ) are rare hematologic diseases characterized by the clonal proliferation of mature blood elements from several myeloid lineages , associated in certain cases with bone marrow fibrosis , splenomegaly , and/or hepatomegaly . they include chronic myelogenous leukemia ( cml ) , three related entities named polycythemia vera ( pv ) , essential thrombocythemia ( et ) , and primary myelofibrosis ( pmf ) ( called philadelphia chromosome - negative ( phi - negative ) mpns ) , chronic eosinophilic leukaemia , mastocytosis , and unclassifiable mpns . cml and other mpns are classified based on the presence or the absence of the bcr - abl fusion gene which is the hallmark of cml . three types of molecular markers are associated with phi - negative mpns : activating mutations in the jak2 gene ( jak2-v617f being the most frequent mutation , present in all subtypes of mpns ) ; activating mutations in the mpl gene ( mpl - w515l / k mostly ) ; and alterations of calr , the gene coding calreticulin ( calr ) , detected in et and in pmf [ 311 ] . a small percentage of mpn patients ( < 15% ) do not carry mutations in the jak2 , mpl , or calr genes . the exact roles played by jak2 , mpl , and calr mutations in the pathogenesis , phenotype , and complications of the three mpn subtypes are not fully elucidated . none of the jak2-v617f , mpl - w515l / k , or calr mutations is specific of a particular mpn subtype . they are detected in patients with very different phenotype and disease evolution , and therefore their presence alone is not sufficient to explain the clinical presentation and complications observed in mpn patients . moreover , for subsets of patients , the jak2-v617f mutation has been shown to be a rather late event , sometimes recurrent , which indicates that other genetic events are responsible for clonality in these patients [ 1418 ] . interestingly , some of the clinical symptoms and complications appear to be linked to the chronic inflammation which almost always accompanies mpn disease , and reduction of symptoms linked to inflammation is beneficial to patients [ 19 , 20 ] . presently it is unclear whether the inflammation - related biological markers and clinical symptoms observed in mpn patients are consecutive or reactive to , or perhaps even precede , the main mutations harbored by mpn clones . obviously , a better understanding of the mechanisms that underlie inflammation in the different mpn subtypes should have a significant impact on the design of future protocols tested for the therapy of mpns . to help address this issue , the present review describes the role played by somatic as well as germline genetic defects in the increased production of inflammatory cytokines and other inflammation markers in mpns ; potential nongenetic causes of chronic inflammation are also discussed . inflammation is a pathological process typically triggered by an external aggression , which may be a physical or chemical injury , irradiation , or infection . in addition , chronic hypoxia ( e.g. , when cells accumulate in a solid tumor or in the bone marrow in the context of blood malignancy or in any type of tissue in case of venous or arterial thrombosis ) can also lead to inflammation [ 2123 ] . chronic inflammation is characterized by the prolonged stimulation of the production of immune blood cells from the lymphoid and myeloid lineages and the release of various mediators , notably inflammatory cytokines , in blood vessels and in tissues . myelopoiesis is stimulated during inflammation so as to produce sufficient quantities of polyclonal granulocytes , monocytes , and macrophages to ensure the destruction of damaged cells , tissues , or infectious pathogens , adequate phagocytosis , and presentation of antigens to lymphocytes . the production of polyclonal megakaryocytes and platelets is frequently increased , to ensure thrombus formation and hemostasis in case of damaged blood vessels in inflamed tissues . chronic inflammation may lead to hypoxia of variable severity in the damaged tissues and , accordingly , to increased production of polyclonal erythroid progenitors and red blood cells in an effort to improve cell and tissue oxygenation . conversely , hypoxia can lead to increased production of inflammatory cytokines : individuals with mountain sickness present with elevated levels of inflammatory cytokines in peripheral blood , and healthy volunteers exposed to a hypoxic environment ( three nights in high altitude above 3400 meters ) presented with a high level of interleukin- ( il- ) 6 [ 24 , 25 ] . patients with chuvash polycythemia associated with homozygous germline mutation in the von hippel - lindau ( vhl ) gene , a major actor of the hypoxia sensing pathway , present with elevated levels of tumor necrosis factor- ( tnf- ) and interferon- ( ifn- ) . inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis also provide evidence of cross talk between hypoxia and inflammation . in rheumatoid arthritis , hypoxia - inducible factor- ( hif- ) 2 is the hif isoform that plays a major role in inflammation , notably by inducing expression of il-6 and tnf- . importantly , hif-1 plays an essential role in survival and function of myeloid cells during inflammation . if the initial injury persists , the inflammation response and associated chronic stimulation of hematopoiesis are prolonged , and the risk of dna alteration increases in cells from the damaged tissues or / and in overstimulated hematopoietic progenitors . over time the acquisition of genetic defects in the inflamed tissues or / and hematopoietic progenitors may eventually lead to the development of solid cancer or / and clonal hematopoiesis and hematological malignancy ( figure 1 ) . in fact , all types of solid and blood cancers , including mpns , are accompanied by some degree of chronic inflammation [ 21 , 22 ] chronic inflammation is an early event in many types of cancers and in certain lymphoma but in mpns , the possibility that chronic inflammation precedes the acquisition of the main mpn mutations is a new subject of research . whatever its chronology , chronic inflammation facilitates further dna alteration in cancer and adjacent cells , and targeting inflammation and its causes should offer new opportunities of cancer treatment and also help reduce complications [ 2123 ] . in the context of solid cancer , chronic inflammation may be reactive to a persistent tissue injury ( exposure to toxics or to infectious agents ) or / and to the tumor itself ; it may also be a consequence of tumor - associated mutations or of treatment ( radiotherapy or chemotherapy ) ( figure 2 ) . thus inflammation may precede or / and accompany malignancy , and polyclonal hematopoietic cells of the myeloid and lymphoid lineages participate in the inflammation process . whatever the cause(s ) of inflammation , sustained stimulation of the proliferation of lymphoid or myeloid cells to maintain inflammation over months or years increases the risk of dna alteration in these cells and the subsequent emergence of a mutated clone ( initiation of malignancy ) or of additional mutated clones ( during or after radio- or chemotherapy ) . figure 2 represents progression from chronic inflammation and stimulation of polyclonal myelopoiesis to clonal myelopoiesis , expansion of a mutated myeloid clone , and myeloid malignancy . in mpns , cells from all myeloid lineages may belong to the malignant clone : erythroid cells , megakaryocytes , neutrophils , and monocytes ; b - lymphocytes or / and t - lymphocytes may be mutated too , but only rarely and usually in pmf . in contrast to patients with solid cancer , for whom myelopoiesis is normal and polyclonal , the immune response in patients with mpns includes the mobilization and activity of mutated ( clonal ) myeloid cells as well as of healthy myeloid and lymphoid cells . depending on the small or large size of the mpn clone , the myeloid part of immune and inflammatory responses may be partially or mostly clonal and subsequently mildly or severely defective . this side of myeloid malignancy is often neglected but likely important in the pathogenesis and complications of mpns . one cause of chronic inflammation recognized as increasing the risk of malignant transformation of affected cells and tissues is chronic infection . indeed it is now well established that latent infection can be associated with various types of solid cancer or / and with lymphoid malignancy [ 3137 ] . in blood malignancies , two main transforming mechanisms may be at play : direct cell infection and transformation by oncogenic molecules or indirect transformation via chronic antigen stimulation and cell proliferation resulting in increased risk of acquisition of genetic defects . during inflammation , cytokines are released which signal cells such as t - lymphocytes and monocytes - macrophages to travel to the site of injury . in turn , activated immune cells increase their production of inflammatory cytokines , chemokines , hematopoietic cytokines , and other growth factors , hereby stimulating numerous cell types from their environment ( fibroblasts and endothelial cells ) , which further increases the production of inflammatory cytokines . in this context , the nuclear factor kappa - b ( nf-b ) and jak1/stat1 pathways are the two main molecular pathways activated to enhance the production of inflammation cytokines ( figures 3 and 4 ) [ 12 , 21 , 38 ] . in case of inflammation linked to hypoxia , which may occur after thrombosis or because of cell accumulation , the production of inflammatory cytokines and growth factors by the cells exposed to hypoxia is upregulated via the hif-1 pathway [ 39 , 40 ] . as shown in figure 3 , the nf-b , hif-1 , and jak / stat pathways interact closely . they act in synergy , nf-b activating the hif-1 pathway , which in turn leads to increased activation of several signaling pathways , including jak2/stat5 ( via the production of erythropoietin ( epo ) ) , stat3 ( via inflammation cytokines from the il-6 family or via epo , hepatocyte growth factor ( hgf ) , platelet - derived growth factor ( pdgf ) , and vascular endothelial growth factor ( vegf ) ) , and stat1 ( via type i and type ii inflammatory cytokines ) ( figure 4 ) . moreover , the level of jak activity affects the expression of transcription factors hif-1 and hif-3 [ 13 , 41 ] . in the context of malignancy , the genetic mutations associated with the tumor may or may not induce the production of inflammation cytokines in mutated cells . this aspect is particularly important in the context of blood cancers since the mutated cells are involved in the immune response or / and are major sources of production of inflammatory cytokines . situations where chronic inflammation results from more than one cause are not rare : physical injury and infection , thrombosis and hypoxia , solid cancer and infection , jak2-mutated mpn and thrombosis , and so forth . the degree of activation and overall synergistic action of the three main pathways which control the production of inflammatory cytokines may vary widely , which allows for infinite qualitative and quantitative differences ( figure 4 ) . thus the cytokine profile and degree of overproduction of inflammatory cytokines and other mediators of inflammation are expected to vary from patient to patient , according to the cause(s ) of inflammation , the cell types being stimulated , and the molecular pathways involved . cytokines may be divided into four groups on the basis of their biological functions : ( i ) natural immunity , for tnf- , il-1 , il-6 , il-5 , il-8 , and chemokines ; ( ii ) lymphocyte activation , growth , and differentiation , for il-2 , il-4 , and transforming growth factor- ( tgf- ) ; ( iii ) regulation of inflammation , also for il-4 , tgf- , and il-1 , il-10 , ifn- , and granulocyte macrophage - colony stimulating factor ( gm - csf ) ; and ( iv ) stimulation of leucocyte growth and differentiation , for il-1 , il-3 , il-5 , il-6 , granulocyte - csf ( g - csf ) , macrophage - csf ( m - csf ) , and gm - csf . cytokines are also classified as th1 ( proinflammatory ) cytokines ( il-1 , il-2 , il-12 , tnf- , and ifn- ) and th2 ( anti - inflammatory ) cytokines ( il-4 and il-10 , notably ) . th1 cytokines cause stimulation of cd8-positive cytolytic t - lymphocytes , leading , for instance , to viral clearance . hence the cytokines produced during chronic inflammation vary according to the cause of inflammation and the cell types involved . the cytokines produced in large quantities during inflammation may also vary according to the molecular pathways that are being activated ( due to the acquisition of mutation(s ) , infection , hypoxia , etc . ) . the cytokines produced following activation of the nf-b and jak1/stat1 pathways include il-1 , il-6 , il-8 , il-10 , il-11 , il-12 , il-13 , il-15 , il-22 , vascular endothelial growth factor ( vegf ) , tnf- , tgf- , platelet - derived growth factor- ( pdgf- ) bb , b - fibroblast growth factor ( fgf ) , g - csf , gm - csf , ifn- , macrophage inflammatory protein- ( mip- ) 1 , mip-1 , mip-3 , hgf , ifn--inducible protein 10 ( ip-10 ) , monocyte chemotactic protein- ( mcp- ) 1 , monokine induced by ifn- ( mig ) , and regulated on activation , normal t - cell expressed and secreted ( rantes ) [ 13 , 40 , 41 ] . in case of hypoxia , increased hif-1 expression leads to the upregulation of the production of epo , vegf , insulin growth factor 2 ( igf-2 ) , tnf- , tgf- , pdgf , fibroblast growth factor ( fgf ) 2 , il-6 , hgf , and its receptor met ( list not exclusive ) . most inflammation cytokines activate the jak1/stat3 pathway , thus ensuring enhanced survival of many cell types , including fibroblasts , endothelial cells , and hematopoietic progenitors . certain cytokines and growth factors activate other molecular pathways , such as the smad proteins for tgf- , jak1/stat1 for ifn , or the jak2/stat5 pathways for epo and g - csf , which stimulate the production of red blood cells and granulocytes , respectively [ 4346 ] . in mpns , several inflammatory cytokines and growth factors ( il-6 , il-8 , gm - csf , hgf , vegf , b - fgf , and tgf- ) are found to be significantly overproduced in all subtypes , yet with a large variability in quantity ( table 1 ) . of note , tgf-1 inhibits normal hematopoiesis in humans via its receptor ii ( tgf-rii ) . in cancer cells , a reduced expression of tgf-rii is frequent , which suggests that malignant mpn progenitors may also acquire resistance to tgf-1 by downregulating tgf-rii expression [ 47 , 48 ] . for certain cytokines , excess production of il-4 , il-10 , and tnf- has been reported in et ; elevation of il-11 levels has been described only in pv ; and in pmf , many cytokines , growth factors , and chemokines are produced at high levels but ifn- levels are usually low ( table 1 ) [ 45 , 4954 ] . the cellular sources of production of inflammation cytokines , chemokines , and growth factors are many and of course vary depending on the mpn subtype and associated complications ( thrombosis and bone marrow fibrosis ) . however , they usually include most of the cell types which constitute the bone marrow microenvironment or hematopoietic niche , fibroblasts , macrophages , t - lymphocytes , and endothelial cells , as well as healthy or mutated ( clonal ) hematopoietic progenitors and mature blood elements , platelets , neutrophils , monocytes , and macrophages [ 5559 ] . macrophages may present with a m1 phenotype , where they produce large amounts of tnf- and il-12 ( both elevated in pv and pmf ) as well as il-23 . macrophages of the m2 phenotype secrete il-4 or il-10 ( both elevated in et ) . in mpns the presence of the activating jak2-v617f mutation in > 95% of pv cases likely compensates for the low epo production by rendering erythroid progenitors highly responsive to low doses of epo , to result in polycythemia . this explanation is likely also valid for the 5060% of et and pmf which are jak2-v617f - mutated . intriguingly , blood levels of other cytokines which also activate the jak2/stat5 pathways ( tpo and g - csf ) or the jak2/stat3 pathways ( gm - csf , il-12 , il-33 , and cytokines of the il-6 family : il-6 , il-11 , and oncostatin m ( osm ) ) are often normal or elevated in mpns ( table 2 ) . several of these cytokines are produced by nonhematopoietic cells and also by myeloid progenitors , and they promote the survival and proliferation of both clonal and nonclonal myeloid progenitor cells . this is the case notably for tpo , il-6 , il-8 , il-11 , il-33 , gm - csf , hgf , and tnf- and several of these cytokines have been proven to contribute to the expansion of the jak2-v617f - mutated cells [ 5153 , 59 , 61 ] . regarding tpo , it is important to note that the low surface expression of mpl ( the tpo receptor ) observed in mpn progenitors and platelets likely limits the effect of high circulating levels of tpo . the reasons for the low expression of mpl in mpn patients are not fully understood . on one hand , jak2-v617f is thought to be less efficient than wild type jak2 to bring mpl receptors to the cell surface and possibly to increase mpl destruction . on the other hand , a high tpo level and activating jak2-v617f or mpl - w515l / k mutations may be ways of counteracting mpl repression in progenitor cells . il-33 is an alarmin known to help fight viral infection that is implicated in autoimmunity , and an increased risk of autoimmune disease has been reported in mpn patients [ 61 , 62 ] . chronic stimulation by the above cytokines also facilitates the survival and expansion of fibroblasts and fibrosis ( il-6 and b - fgf ) , monocytes - macrophages ( il-6 and gm - gsf ) , and platelet production ( il-6 ) and neoangiogenesis ( vegf ) , whereas il-12 and il-33 activate t - lymphocytes and natural killer ( nk ) cells . in addition , mpn cells accumulate in the bone marrow , which leads to some degree of hypoxia and subsequent activation of the hif-1 pathway , with upregulation of stat3 expression and production of cytokines which further promote cell survival ( igf-2 , hgf , and il-6 ) , fibrosis ( pdgf , fgf2 , and il-6 ) , and neoangiogenesis ( vegf ) [ 42 , 63 ] . altogether , the qualitative and quantitative differences found in cytokine production in the three mpns subtypes hint that the causes and mechanisms of chronic inflammation likely differ in et , pv , and pmf . the jak2-v617f , mpl - w515l / k , and calr mutations likely influence clinical symptoms but do not explain differences in inflammation . for instance , jak2-v617f , mpl , and calr mutations are detected at similar levels of expression in et ( associated with mild or very mild inflammation ) and in pmf ( characterized by severe inflammation ) . thus it is important to investigate and understand the mechanisms of inflammation at play in each mpn subtype , including those independent of jak2 , mpl , or calr mutations . the main clinical symptoms observed in mpns which are linked to an increased production of inflammation cytokines are fatigue , fever , itching , night sweats , weight loss , and , to some extent , splenomegaly . these symptoms are frequent in pmf ; they occur in pv but are mostly absent in et , which is a good reflection of the degree of production of inflammation cytokines characteristic of pmf ( high or very high ) , pv ( moderate to high ) , and et ( mild ) . the main biological parameters routinely assessed which are affected in case of inflammation include blood cell counts ( in particular leukocyte , neutrophil , and platelet counts ) , iron levels , and several proteins : the c - reactive protein ( crp ) , haptoglobin , alpha-1 acid glycoprotein ( orosomucoid ) , ferritin and fibrinogen ( increased ) , and albumin and transferrin ( decreased ) . these inflammatory proteins present with different kinetics : inflammatory positive markers which are increased early include crp , haptoglobin , and alpha-1 acid glycoprotein , whereas fibrinogen , ferritin , and transferrin are late - acting inflammatory proteins . elevation of the leukocyte and platelet counts is typical of mpns and thus does not allow distinguishing between inflammation and mpn . crp is elevated in mpns , particularly in pmf , and pentraxin 3 has been reported to decrease in mpns . a high crp and low pentraxin 3 were linked to a high risk of thrombotic events in pv and in et , and a high crp was associated with shortened leukemia - free survival in mpn patients with myelofibrosis [ 64 , 65 ] . the level of il-6 in serum is almost constantly increased in case of mpn but il-6 levels ( and other inflammatory cytokines ) are not measured in routine laboratory practice . mpns are characterized by the activating jak2-v617f and mpl - w515l / k mutations , the calr mutations , and also high levels of total jak2 ( wild type and v617f - mutated ) in neutrophils and platelets [ 310 ] . the effect of jak2-v617f mutation is to activate primarily the stat5 pathways but the stat3 pathways are also activated ( figure 4 ) . the mpl - w515l / k mutations presumably stabilize mpl , the jak2-coupled dimeric receptor for tpo . tpo is known to stimulate the jak / stat pathways and also pi3k / akt , erk , p38 , nf-b , and hif [ 6769 ] . accordingly , in transfected cells expression of mpl - w515l / k mutants resulted in increased activation of erk ( extracellular signal - regulated kinases ) 1 and erk 2 ( erk1/2 ) and akt ( protein kinase b ) in absence and in presence of tpo [ 5 , unpublished observations ] . to our knowledge , the effect of mpl - w515l / k mutations on nf-b and hif has not been studied . in any case , the jak2-v617f mutation activates stat3 and the mpl - w515l / k mutations activate stat1 and stat3 , which implies that they may stimulate the production of inflammatory cytokines ( figure 4 ) . however , in mpn progenitor cells and platelets , the expression of mpl receptors at the membrane surface is often very low , which likely attenuates the effect of tpo stimulation and mpl - w515l / k mutation . calreticulin is a calcium - binding protein chaperone normally located in the endoplasmic reticulum ( er ) ; the calr mutations associated with mpns all result in c - terminal truncated forms of calreticulin located in the cytosol . thus it is presumed but not formally demonstrated that calr mutants may affect intracellular calcium flux as well as the trafficking and secretion of glycoproteins , which could potentially lead to altered expression and activation of cytokines , receptors , jak2 , and other signaling molecules . consistently , the initial papers reported an activation of the jak2/stat5 pathway in transfected cells which expressed calr exon 9 mutants [ 8 , 9 ] . however , the precise molecular mechanisms linking calr mutants and the jak2/stat5 pathway have not been identified . more rarely , in et or pmf the driving mutation may be a loss - of - function mutation in the lnk gene or in the cbl gene [ 7074 ] . lnk is an adaptor protein which acts as a negative regulator of tpo / mpl - mediated activation of jak2 . expression of lnk loss - of - function mutants also results in enhanced activation of the jak2/stat5 pathway . cbl codes for an e3-ubiquitin ligase which promotes the ubiquitination of signaling molecules , including tyrosine kinases . the cbl mutations detected in mpns cause the loss of e3-ubiquitin ligase activity , thus resulting in increased signaling and cell proliferation . so far there is no evidence that lnk or cbl mutations induce the production of inflammatory cytokines , but they may alter their signaling . mutations in the tet2 , idh1 , idh2 , ezh2 , aslx1 , or dnmt3a genes may also be found in mpns . they are not specific of mpns : they are found also in other blood and solid malignancies . tet2 and idh1/2 mutants impair the hydroxylation of methylcytosine and thus affect dna methylation . more precisely , tet gene products catalyze the conversion of 5-methylcytosine to 5-hydroxy - methylcytosine ( 5-oh - mec ) , a reaction that depends in part on iron and oxygen [ 80 , 81 ] . ezh2 ( enhancer of zeste homolog 2 ) gene codes for a histone methyl transferase , and asxl1 ( additional sex combs like transcriptional regulator 1 ) gene product belongs to the polycomb group of proteins and thus is thought to disrupt chromatin and alter gene transcription . dnmt3a codes for a dna methyltransferase and mutations presumably alter the epigenetic regulation of gene expression . thus one can not exclude that these mutations may alter the expression of genes coding for inflammatory cytokines or receptors . several groups have studied the production of inflammation cytokines in jak2-v617f - mutated cells or in murine jak2-v617f - driven mpn models . so far published reports concluded that , in vitro , jak2-v617f can increase the production of il-6 , il-8 , il-9 , osm , ccl3 , ccl4 , and tnf- [ 53 , 59 , 84 , 85 ] . however , in mpn patients there is no correlation between the jak2-v617f burden and the blood or serum levels of these cytokines . in fact , it is highly probable that only a fraction of these cytokines is under the control of jak2-v617f . firstly , il-6 , il-8 , and osm are abundantly produced by nonhematopoietic ( nonclonal and nonmutated ) cells [ 5153 ] . secondly , certain molecules produced under the control of jak2-v617f , such as osm , in turn stimulate the production of other inflammatory cytokines in a jak2-v617f - independent manner . thirdly , in the jak2-v617f hel cell line , anti - jak2 mirna experiments had only a partial inhibiting effect on il-6 mrna expression ; in these experiments , anti - jak2 mirna experiments had no effect on the expression of il-11 and hgf . thus in jak2-v617f - mutated cells , major inflammatory cytokines may be controlled partially ( il-6 ) or totally ( il-11 and hgf ) by molecular pathways not regulated by jak2-v617f . regarding mpl - w515 mutations , only one group reported the analysis of inflammation cytokines produced in mpl - w515l - mutated cells , in a murine bone marrow transplantation model : expression of mpl - w515l was associated with a significant increase in the production of il-6 , il-10 , il-12 ( p40 ) , tnf- , csf3 , and chemokines ccl2 , cxcl9 , and cxcl10 . again , mpl - w515l - mutated cells were not the sole source of production of these cytokines . regarding the calr exon 9 mutations associated with mpns , their effect on cytokine expression is not known . it is interesting to note that soluble calreticulin has been reported associated with increased production of il-6 and tnf- . regarding tet2 , idh1/2 , and asxl1 mutations , it was reported that mutated forms of idh1/2 were associated with specific dna hypermethylation profiles , and the list of genes found to be differentially methylated includes several genes linked to inflammation , particularly the il11-r and tgf-ri receptors . interestingly , il-11 and tgf- are secreted at high levels in case of inflammation and both alter myelopoiesis . hypermethylation of the genes encoding il11-r and tgf-ri receptors would presumably lower their expression and subsequently make clonal progenitor cells less sensitive to the inhibiting action of tgf- and anti - inflammatory action of il-11 . since tet2 and idh1/2 mutations are mostly found in pmf , it is possible that these mutants play a role in the aggravation of inflammation observed in severe forms of pmf [ 87 , 88 ] . in myelodysplastic syndromes , asxl1 mutations combined with setbp1 mutations were reported to repress the tgf- pathways . however no study of cytokine or receptor protein expression in relation to asxl1 mutation in mpns has been published . germline defects , variants , or haplotypes can affect , directly or indirectly , the expression or signaling of inflammatory cytokines and receptors , thus potentially attenuating or aggravating chronic inflammation . the 46/1 ( jak2 ggcc ) haplotype and single - nucleotide polymorphisms ( snp ) in jak2 , in the telomerase reverse transcriptase ( tert ) , in the mds1 and evi1 complex locus ( mecom ) , or in hbs1l - myb have been reported to be associated with a predisposition to mutation in the jak2 gene on the same allele ( jak2 ggcc haplotype ) or a predisposition to the development of a mpn ( mecom , tert , jak2 , and hbs1l - myb variants ) [ 9094 ] . to this day it remains unclear how these hereditary genetic variants act to facilitate the development of mpns , but alteration of the transcription of the concerned genes is possible . regarding germline jak2 variation , inappropriate expression of jak2 would clearly disturb myelopoiesis and alter the contribution of myeloid cells to inflammation responses . consistently , the jak2 ggcc haplotype was reported to be associated with a defective response to cytokine stimulation , increased risk of inflammation , and impaired defense against infection [ 95 , 96 ] . in cml , cells with short telomere length were found to express a specific telomere - associated cytokine and chemokine secretory phenotype . little is known on the functional effects of mecom variants on cytokine production but yasui et al . recently reported that the evi1 oncoprotein could alter tgf- signaling and tgf--mediated growth inhibition [ 98 , 99 ] . it is established that variations due to snps in the promoter region of genes coding for inflammatory cytokines and receptors potentially affect their production . many groups have published snps associated with an altered production of a cytokine or a cytokine receptor , and such snps concern all the main cytokines involved in inflammation : il-1 , il-1r , il-2r , il-6 , il-8 , il-10 , il-12 , il-33 , tnf- , hgf , and mcp1/ccl2 [ 100115 ] . snps have been shown to control the expression of these cytokines in vitro and individuals who carry the snp are described as high or low producers [ 116118 ] . cytokine polymorphisms have been studied in association with specific diseases , response to infectious agents , or immune response to inflammation . chronic inflammation associated with mpn may have several causes , and their recognition should allow offering improved and individualized treatment to mpn patients . as described above , part of the inflammation is clonal since mpn clonal cells produce inflammatory cytokines ( il-6 , il-8 , il-9 , il-11 , osm , tnf- , ccl3 ( mip-1 ) , and ccl4 ( mip-1 ) ) ; the eventual acquisition of idh1/2 or tet2 mutations may aggravate clonal inflammation by altering the expression of certain receptors ( il-11r , tgf-r1 ) . the consequences are multiple : ( i ) enhanced survival and growth of clonal cells ( il-6 , il-8 , il-11 , and tnf- ) ; ( ii ) increased production of inflammation cytokines that target bone marrow stromal cells as well as hematopoietic progenitors , via the action of osm , il-11 , and il-6 ; ( iii ) resistance of clonal cells to growth inhibitors , via a reduced expression of il-11r or tgf-r1 [ 53 , 59 , 61 , 63 , 119 , 120 ] . in addition , clonal cells can recruit and activate neutrophils , monocytes , and natural killer cells via the production of ccl3 and ccl4 ; the neutrophils and monocytes potentially recruited may be clonal or non - clonal . any malignant process induces nonclonal immune responses which aim to restrict and eventually destroy the malignant cells . in case of advanced disease , nonclonal inflammation may also be reactive to treatment . in mpns , the mature myeloid cells which participate in the antitumoral or hypoxia - induced or therapy - related reactive inflammation response may be clonal or nonclonal . depending on the mpn subtype , the size of the clonal population is likely to be large ( pv and pmf ) or moderate or small ( et ) , implying that the clonal part of reactive inflammation is probably more significant in pmf and pv than in et . this should not be overlooked because clonal cells likely mount a less efficient immune response than healthy cells , meaning that the inflammation / immune response could be rather inefficient in pv and pmf . the observation that major inflammatory cytokines are produced independently of mpn - associated mutations and the demonstration that jak2-v617f can be a late event in mpn development are consistent with the hypothesis that chronic stimulation of myelopoiesis ( via inflammation ) may precede the acquisition of mutation in the jak2 ( mpl and calr ? ) a frequent objection is the lack of evidence of inflammation or myeloid stimulation prior to the diagnosis of a mpn . however , it is not rare that routine blood tests of patients , especially older patients , reveal a slight elevation of leukocyte or platelet counts , or hematocrit , with or without biological evidence of mild inflammation . there are dozens of reasons for mild alterations of blood counts , ranging from smoking , stress , obesity , and diverse latent infections to mild forms of chronic inflammatory diseases ( intestinal , rheumatoid , skin , type 2 diabetes , atherosclerosis , etc . ) . such patients are simply observed ; investigation begins when blood counts rise significantly ( reach at least one of the who criteria of mpn ) or when patients present clinical symptoms related to mpn or to the underlying cause of chronic myeloid stimulation or inflammation . also , it is not rare to detect lymphoid infiltrates in the bone marrow of mpn patients and monocytosis or lymphopenia in peripheral blood , sometimes prior to the diagnosis of mpn ; these observations may be considered as evidence of a disturbed immune response . thorough investigation of the stages preceding the diagnosis of overt mpn , similar , for instance , to the studies that established monoclonal gammopathy of undetermined significance ( mgus ) as the precancerous stage of multiple myeloma , is needed in mpns to validate the hypothesis of chronic ( antigen - mediated or not ) stimulation of myelopoiesis preceding the acquisition of jak2 , mpl , or calr mutation . the chronic inflammation and myeloid stimulation hypothesis is attractive , because it can explain several if not all of the mysteries that persist in mpns . for instance , chronic myeloid stimulation allows the recurrent acquisition of jak2-v617f , multiple jak2 mutations , and combinations of jak2 , mpl , or calr mutations regularly reported in mpns . early chronic inflammation and myeloid stimulation would explain that jak2-v617f burden and clinical symptoms and disease severity are not correlated . the recent reports that patients under treatment with jak inhibitors may develop or reactivate viral infection , possibly due to impaired nk cell function , are also consistent with chronic infection contributing to the inflammation associated with mpns . importantly , the chronic stimulation hypothesis allows for multiple causes of inflammation , infectious or not , some mild ( as observed in et ) and some severe ( as typical of pmf ) . last but not least , the chronic myeloid stimulation hypothesis allows for many different initial causes and thus would explain why the jak2-v617f mutation and to a lesser degree the mpl exon 10 and calr exon 9 mutations are associated with very different diseases ( et , pv , pmf , rars - t , and splanchnic thrombosis for jak2-v617f ; et , pmf , and rars - t for mpl and calr mutations ) . for all these reasons , chronic myeloid stimulation and inflammation , and notably latent infection , deserve investigation as initial , early , or complicating events of mpns . indeed chronic exposure to various toxic compounds or to infectious pathogens and subsequent chronic inflammation frequently precedes malignant cell transformation in the context of solid cancers ; importantly , the same toxics or infectious agents associated with solid cancer may also lead to lymphoid malignancy ( table 2 ) [ 3137 ] . normal immune responses following infection include the stimulation of myelopoiesis ( granulocytes and monocytes ) . the production of b - lymphocytes and plasma cells is stimulated to produce polyclonal ig . if infection becomes chronic , a focusing of the ig response from polyclonal to monoclonal ( mc ) immunoglobulins ( ig ) may occur , and that will persist as long as the infection . epstein - barr virus ( ebv ) , hepatitis c virus ( hcv ) , hepatitis b virus ( hbv ) , or helicobacter pylori ( h. pylori ) stimulate polyclonal b - cell proliferation , and these pathogens are implicated in several b - cell malignancies ( burkitt , hodgkin , non - hodgkin lymphoma , and chronic lymphocytic leukemia ) via cell infection and direct transformation ( ebv and hcv ) , via antigen - driven stimulation ( h. pylori ) , or both ( ebv and hcv ) [ 3237 ] . moreover , ebv , cytomegalovirus ( cmv ) , hhv-8 , and hhv-6 can induce a chronic monoclonal ig response [ 123126 ] . in support of the hypothesis that infection may predispose to chronic hematological malignancy , we showed that , for about 25% of patients with multiple myeloma , the purified mc ig specifically recognizes an antigen from hcv , ebv , or h. pylori [ 124127 ] . these important findings suggest that infectious agents may also initiate multiple myeloma , not just certain types of lymphoma , which opens new possibilities of curative treatment , as demonstrated recently by the regression of one case of hcv - associated myeloma following treatment by ifn- . antigen - driven proliferation as a facilitator of dna mutation acquisition and cell transformation is rarely investigated in the context of myeloid malignancies but since chronic antigen stimulation also concerns myeloid cells , latent infection as a cause of inflammation in chronic myeloid disorders should not be excluded . thus a promising research approach for chronic myeloid disorders is to propose that , for subsets of patients , malignancy may result from chronic , polyclonal abnormal immune response by myeloid cells , eventually facilitating excessive myeloid proliferation , acquisition of genetic alterations in genes that are critical for myelopoiesis ( jak2 and mpl ; calr ? ) , and transformation of progenitor cells from the most stimulated lineage(s ) and then expansion of a malignant clone . logically , the jak2-v617f mutation rapidly became the main target of treatment in mpns after its discovery in 2005 . in contrast , chronic inflammation has so far been neglected in the treatment of these diseases . recognition of the importance of inflammation in the pathogenesis of mpns offers great opportunities to improve therapy . the jak inhibitor trials showed that blocking jak2 function significantly reduced inflammatory cytokine levels and other markers of inflammation in pmf patients , resulting in improved clinical symptoms . patients benefited from jak inhibitors even when the jak2-v617f mutant burden was not reduced or when their disease was not associated with jak2 mutation . although the comprehension of the causes and mechanisms of inflammation in mpns is still very incomplete , accumulated knowledge indicates that nf-b and jak1 are major pathways for the production or / and signaling of inflammatory cytokines . in certain cases , the three pathways are closely linked ( figures 3 and 4 ) , and used alone , inhibitors of the jak / stat pathways ( or inhibitors of nf-b ) can not be expected to completely block cytokine production and signalling in mpns . in a murine model of jak2-v617f - mutated mpn , in fact , alteration of stat3 function ( deletion or hyperactivation ) is known to lead to altered myelopoiesis and increased expression of stat1 and inflammatory cytokines , notably il-6 , a strong stimulant of platelet production , fibroblast proliferation , and inflammatory acute phase protein production [ 130132 ] . in support of this mechanism , grisouard et al . reported increased expression of stat1 and stat1 target genes in jak2-v617f mice after stat3 deletion ; il-6 and other inflammatory cytokines were not measured in this study . ideally to cure a mpn , one should aim to reduce the effects of the jak2 , mpl , or calr mutant carried by the mpn clone , as well as the production and signaling of the main inflammation cytokines produced by the patient . this can be achieved by blocking the three main pathways responsible for cytokine production ( these include the jak1/jak2 pathways ) and by suppressing the cause(s ) of mpn mutation , when identified . used alone , jak1/2 inhibitors have the capacity to block the jak2-v617f and mpl - w515l / k mutations and a large fraction of the production and signaling of inflammatory cytokines . but for complete treatment of inflammation and mutations in mpns , the addition of nf-b and hif-1 inhibitor drugs should benefit patients [ 133138 ] . this contrasts with myeloma , a disease not driven by the activation of the jak2/stat pathways where nf-b and hif-1 inhibitors used alone can reduce both disease and inflammatory cytokines . another advantage of such combination therapies would allow lowering the dose of each drug and hopefully reduce toxicity . of note , one reason why ifn- is able to induce a complete clinical , biological , and molecular remission ( jak2-v617f - negativation ) in pv and in et patients is that ifn- acts on several jak / stat pathways as well as on other pathways critical for the production of inflammatory cytokines [ 140 , 141 ] . in short , as represented in figure 5 , the ideal mpn therapy may combine the following : ( 1 ) inhibition of the jak1 pathway and jak2-v617f , mpl - w515l / k , or calr mutations with a jak1/2 inhibitor and ( 2 ) nf-b and hif inhibitors ( note that ( 1 ) and ( 2 ) may be achieved with ifn- ) . whenever an early cause of chronic inflammation is identified , adequate treatment should be added : for instance , antibiotics or antiviral treatment in case of latent infection . the complexity of inflammation in mpns should not discourage attempts to define it biologically at the time of diagnosis , prior to therapeutic decisions , and during treatment monitoring . knowing the precise inflammation status of each mpn patient would greatly help improve his / her treatment . as described earlier , the inflammation status and cytokine profile of a mpn patient are expected to vary according to the mpn subtype , presence of jak2 , mpl , or calr mutation , eventual cause of inflammation preceding mpn - driving mutation , and personal genetic background . yet what matters for therapy is the resulting cytokine profile of the patient , and nowadays establishing the inflammation cytokine profile of an individual is technically simple and not overly expansive and requires only a blood sample . knowing that a patient is a strong producer of il-6 , hgf , or tnf- , for instance , would allow focusing treatment on the target cytokine(s ) , perhaps by adding to the patient 's combination therapy one of the existing antagonist drugs or neutralizing antibodies that specifically block these cytokines or receptors [ 119 , 142144 ] . last but not least , extensive genetic studies and murine models have not succeeded to fully explain most of the chronic hematological malignancies , including mpns . this suggests that genetic aberrations , although crucial , are probably not sufficient for a lymphoid or myeloid malignancy to develop , and more attention is now given to the hematopoietic niche and cytokine production , the human microbiome and oncogenic infectious pathogens , and the host 's immune response [ 145 , 146 ] . there is no reason to limit these important pathogenic mechanisms to lymphoid malignancies and solid cancer , and perhaps the next major research effort in the mpn field should be to investigate the validity of the hypothesis of chronic inflammation / myeloid stimulation preceding mutation acquisition . more specifically , a systematic search for latent infection in mpn patients is feasible and simple , thanks to various tests based on the multiplexed antigen or peptide microarray technology ; these assays require only a small blood sample [ 127 , 147 ] . obviously the identification of an infectious cause of inflammation in subsets of mpn patients would offer additional possibilities of combined treatment ( with antibiotics or antiviral therapy ) ( figure 5 ) . regarding research and animal models of mpns , it is possible to develop new murine models of chronic myeloid stimulation , antigen - mediated or not . in conclusion , inflammation is very complex yet there are relatively simple laboratory tools to diagnose and characterize inflammation in patients . several predictive inflammation markers are already identified in mpns , and potent drugs that target the molecular pathways of inflammation or the inflammatory cytokines detected in excess in patients already exist . designing new , more complete , and individualized combination treatments that include drugs that block mpn mutations as well as charcot neuropathic osteoarthropathy ( cn ) is a chronic , progressive condition of bones , joints , and soft tissues , most commonly occurring in the area of the foot and ankle as a result of peripheral neuropathy . it is characterized by a local inflammatory process in the early stages and gradual development of bone loss , joint dislocation , and fixed deformities . these deformities diabetes mellitus , together with neuropathy , is currently considered the main cause of cn . data indicating the prevalence and incidence of the condition suggest that it often goes undiagnosed among sufferers of diabetes , with figures ranging from 0.4 to 13% among diabetics . however , changes diagnosed by x - ray and corresponding with cn are detected in up to 29% of diabetics . bilateral disability has been observed in numbers ranging from 9 to 39% of patients . when mri is used as a diagnostic method , the common issue is an early diagnosis and an appropriate treatment , in case of an acute phase where it is difficult to differentiate an acute osteomyelitis . even though the treatment of cn is mostly conservative however , the crucial question is when , where , and how a surgical therapy has to be used . a pubmed search was done with the key word charcot foot , neuropathic arthropathy , charcot arthropathy . we could trace about 400 up - to - date papers on the subject . electronic database was systematically searched for literature discussing the history , pathophysiology , assessment , imaging methods , diagnosis including osteomyelitis , classification , and management of cn . reasons for exclusion of articles based on title or abstract were ( 1 ) nonoriginal data ( e.g. , editorials , guidelines , and comments ) , ( 2 ) nonclinical articles ( e.g. , technical or animal studies ) , ( 3 ) case reports , and ( 4 ) articles not written in english language . all authors independently chose the most up - to - date papers with regard to target topics resulting in the identification of 59 most pertinent articles . together we discussed and compared the relevant information from all these sources with our clinical practice and included them in this review . later , mitchell supposed the relation between spinal lesion and rheumatism of lower extremities in 1831 . charcot described the neuropathic aspect of the condition in detail in 1868 and detected spinal damage resulting from tabes dorsalis as a cause and his brilliant presentation , demonstration of arthropathic affections of locomotor ataxy , at the 7th international medical congress ( 1881 ) , established this disease as a distinct pathological entity . much later , in 1936 , jordan revealed diabetes mellitus to be a possible cause of neuropathic osteoarthropathy . nevertheless , the etiology , diagnostics , and treatment of this condition have to this day yet to be fully addressed . two main theories concerning the origin of the condition have been discussed in the past . the neurotraumatic theory is based upon damage to sensory feedback resulting from progressive destruction of bones and joints brought about by repeated trauma . the neurovascular theory highlights the changes in blood supply caused by neuropathy , most of all lesions in the sympathetic nerves which affect bone resorption . the current accepted theory of cn origin states that , in susceptible individuals with peripheral neuropathy , an unregulated inflammatory process is triggered which leads to an increased expression of the polypeptide receptor activator of nuclear factor kappa - b ligand ( rankl ) . rankl triggers the synthesis of the nuclear transcription factor , nuclear factor- ( nf- ) , and this in turn stimulates the maturation of osteoclasts from osteoclast precursor cells . at the same time , nf- stimulates the production of the glycopeptide osteoprotegerin ( opg ) from osteoblasts . a repetitive trauma with the loss of pain sensation results in continual production of proinflammatory cytokines , rankl , nf- , and osteoclasts , which in turn leads to continuing local osteolysis . another possible cause is decreased secretion of the calcitonin gene related peptide ( cgrp ) from damaged nervous endings . under physiological circumstances this peptide works as an antagonist of the rankl synthesis and at the same time the powerful bone anabolic wnt/-catenin pathway plays a critical role in remodeling as well as preservation of the foot skeleton in the acute and chronic stages of the disease . in general , diabetes may predispose to cn occurrence through a number of mechanisms . apart from the presence of neuropathy and possible osteopenia , these include the effects of advanced glycation end products , reactive oxygen species , and oxidized lipids , which may all enhance the expression of rankl in diabetes . the diagnosis is based on patient 's history , clinical examination , and imaging methods . patients are quite often not aware of any injury as a result of their lowered perception of pain . trigger factors of development of the cn can also be infections ( osteomyelitis precedes cn ) and revascularization . a necessary condition for the emergence of cn is the presence of peripheral neuropathy , in particular , diabetic distal sensitive polyneuropathy . the most common is the semmes - weinstein 5.07/10 g monofilament . next is the pinprick test or even the more sensitive neurometer test . a small fiber - predominant neuropathy is an early manifestation of diabetic neuropathy and it can progress to distal symmetric polyneuropathy . it can be difficult to diagnose because the examination ( decreased reflexes , impaired vibration , and weakness ) and electrodiagnostic testing can be normal . autonomic neuropathy ( a type of small fiber neuropathy ) is also common and plays a key role in the development of the cn . symptoms include gastroparesis , constipation , urinary retention , erectile dysfunction , and cardiac arrhythmias . signs of inflammation are an important sign since inflammation plays a key role in the pathophysiology of cn . oedema , erythema , warmth , and more than a 2c difference in local temperature in comparison to the contralateral extremity are typical symptoms of active cn and it could be difficult to differentiate them from phlegmon with osteomyelitis or from an acute attack of gout , especially since pain occurs in only 50% of neuropathy cases . in this stage the vascular supply to the foot is still maintained though it could be difficult to palpate an arterial pulse due to prominent swelling . the eventual fracture and joint dislocation can lead to deformities , typically to rocker bottom foot with possible ulceration . during this time skin temperature measurement is the most widely used method in the assessment of the activities of the cn . with the use of a surface - sensing temperature device ( infrared thermometer ) , temperatures are recorded in the most affected area of the foot and compared with the same areas of the contralateral foot . for further prognosis and therapy instability of the forefoot can be assessed according to assal and stern : the pressure on the foot in the sagittal plane dorsally when the ankle joint is locked in dorsiflexion . relatively common in cn is the contraction of the triceps surae muscle which is involved in plantar inclination of the calcaneus ( figure 1 ) . primarily it must be emphasized that the changes on the x - ray are typically delayed and have low sensitivity . a basic examination is an x - ray of the talus and the weight bearing foot in the anteroposterior and dorsoplantar lateral projection . during the initial stage the x - ray finding can be negative or only minor bone infractions and joint incongruence are present . in a developed stage fractures and subluxations or luxations the x - ray finding depends on the specific type of cn . in a typical rocker bottom deformity this deformity arises due to deformed midtarsal bones and the shortening of the achilles tendon , which loses its elasticity during glycosylation . another finding on the lateral projection due to deformity of the middle part of the tarsus is a negative angle between the axis of talus and i metatarsus ( figure 2 ) . the dorsoplantar projection shows changes of the position in the lisfranc as well as chopart joint , with resulting abduction or adduction deformity of the foot . examination using magnetic resonance imaging is a very valuable method for the early stages of the illness when x - ray imaging alone results in practically normal findings . an important finding is oedema of the bone marrow of two or more bones , oedema of the adjacent soft tissues , and fluid in several joints or cortical fractures . certain methods of nuclear medicine can be helpful not only as an alternative diagnostic method , for example , in revealing the presence of osteomyelitis , but also for monitoring the progress of the treatment . three- or four - phase bone scintigraphy ( tc - mdp ) is highly sensitive but with a low specificity . scintigraphy with labelled leucocytes ( tc - wbc nebo in - wbc ) is highly sensitive and very specific for diagnosing the infection but it is difficult to differentiate soft tissue from bone . that is why either a combination of both the abovementioned methods or fdg - pet / ct is recommended . the diagnostics of osteomyelitis in cn is difficult primarily in the active stage of the disease when clinical symptoms are practically the same both in osteomyelitis and in cn . on the contrary , in the chronic stage symptoms in osteomyelitis due to ischaemia and immunodeficiency diagnosing osteomyelitis is not possible on the basis of one examination alone . in general , for the differential diagnosis of osteomyelitis and cn , an important role plays a complex view of inflammatory markers and clinical manifestations of infection . the origin of osteomyelitis is in most cases caused by spread of infection from the soft tissues . history of ulceration or presence of ulceration and/or previous amputation in the area of the foot are possible factors to weigh when suspecting osteomyelitis . a high predictive value for the presence of osteomyelitis is ulcerations bigger than two cm and deeper than three mm . the basis of the test is whether a fine blunt steel probe can penetrate the ulceration to the bone . laboratory readings in cn do not show higher markers of inflammation ( practically normal numbers of leucocytes , crp , procalcitonin , and fw ) . due to the chronicity of infection in osteomyelitis we can find particularly higher fw ( most commonly > 70 mm / hour ) . these changes are visible within two to three weeks of the onset of the infection . the abovementioned methods of nuclear medicine ( four - phase bone scintigraphy , marked leucocytes , f fdg - pet , or ga spect / ct ) can also be used to diagnose osteomyelitis , and the range of osteomyelitic changes is also highly visible on the mri . also tc - wbc spect / ct hybrid image is a useful tool in addition to mri . the most reliable examination is a bone biopsy , either preoperative percutaneous or peroperative , made with a biopsy needle after disinfection of the ulceration . swabs are often contaminated with normal skin flora or colonizers , and their use may result in failure to identify deep tissue pathogens . for identification of pathogens , the international working group on diabetic foot ( iwgdf ) however , swab cultures are less invasive than tissue biopsy or curetted tissue and swab culturing ( including a vacuum transport container ) may be reliable for identification of pathogens in superficial diabetic foot wounds . typical ischaemic symptoms like claudication and pains at rest , which would normally appear in the history , might be unrecognised because of the presence of neuropathy . during a physical examination pulsation an example of a noninvasive diagnostic method that can be also used would be measuring blood pressure in the ankle using a doppler probe ( it carries a higher risk of artificially higher pressures in the case of mediocalcinosis ) . in our department we use measure pressure on the big toes or transcutaneous oxygen tension ( this method carries a risk of artificially lower pressures in the case of oedema ) . if angiography is performed postoperatively , there could be a risk of activating cn after prospective revascularization . examination of the vascular supply must be repetitive even after surgical reconstruction of cn when a higher risk of ischaemia exists ( oedema , thrombus formation of vessels ) . based upon our experience , we evaluate this risk as the most significant from the point of view of possible postoperative complications . the most commonly used classification according to eichenholtz was published in 1966 ( table 1 ) . currently , in spite of the quite widespread usage of this classification , it is necessary to consider and search for new alternative methods of imaging . eichenholtz evaluated x - ray images in 68 patients ( altogether 94 joints ) of whom only 12 were diabetic . the development of examination methods of nuclear medicine and magnetic resonance have shown that there are already noticeable and recordable changes of cn even when x - ray images are negative , and starting treatment at such an early stage can prevent deformities . this stage has been labelled as stage 0 and has been added to the original classification . alternatively , stage 1 has been divided into 1a and 1b . with regard to the fact that the initial change in cn is an inflammatory reaction , which corresponds with oedema of bone marrow , a classification of cn based upon mr imaging has been suggested . it recognises two stages of the disease active and inactive according to the presence or absence of oedema of the bone marrow and distinguishes two grades0 and 1according to the presence or absence of cortical fractures . these fractures represent a worse prognosis from the point of view of developing deformities ( table 2 ) . this method is based on immobilisation and the complete absence of weight bearing for the affected extremity in the active stage . there are various opinions concerning the type of immobilisation and the period of nonweight bearing for the foot . the most common immobilisation used is a total contact cast ( tcc ) changed three days after the initial application and then every week . alternatively , it is possible to use charcot restraint orthotic walker ( crow ) prefab orthoses . the period of fixation depends on the reduction in oedema and a drop in skin temperature below 2c compared to the contralateral extremity . the recommended length of fixation varies from six weeks to three months followed by a change of orthosis . similarly , the recommended period without any weight bearing varies starting from weight bearing during application of tcc to the usage of a wheelchair as a preventive means against overloading the other extremity . recommend six to eight weeks of tcc and a wheelchair with subsequent change for individual orthosis fixing the affected segment and at the same time preventing tibial rotation , thus enabling only axial weight bearing ( a so - called frame orthosis ) . in the chronic stage of the condition , a deformed foot in plantigrade position capable of weight bearing in shoes or an orthosis without increasing deformity is suitable for conservative treatment . the type of the prosthetic equipment depends on the gravity of the deformity and on the eventual presence of ulceration . it is possible to use various types of walkers , ankle - foot orthoses , orthotic shoes , or adjusted regular shoes . in general we prefer conservative treatment in both stages by a multidisciplinary team . only after the failure of conservative management does a patient this treatment is beneficial in cn refractory to off - loading and immobilisation or in the case of recalcitrant ulcers . a separate question is the problem of weight bearing in the case of conservatively treated or operated cn . we have the same experience as koller et al . , most of the patients suffering from peripheral neuropathy are not able to reduce weight loading of the foot in a controlled manner with the help of crutches , and there is a risk of overloading the contralateral limb and a risk of fall . therefore we recommend full weight bearing wherein we gradually prolong the time and speed of the walk . to support healing some medicaments have been used , bisphosphonates , which inhibit osteoplastic bone resorption , and intranasal calcitonin , which has had fewer complications . nevertheless , beneficial effect of pharmacological treatment ( improvement of markers of resorption versus an absence of clinical marks of healing and side effects of the therapy ) as well as physical stimulation of the bone growth is yet to be fully demonstrated . besides conservative treatment , the possibilities of surgical treatment have also been looked into and the benefits and risks of such treatment have been considered . saltzman et al . evaluated retrospectively conservative treatment of 127 extremities in 115 patients over a period of 20 years . the study found that the annual rate of amputations was 2.7% , 47% of patients used an orthosis for a period longer than 18 months , and the risk of ulceration appeared in 40% of patients . ulceration is often accompanied by a high risk of amputation . at present , specific methods for the surgical treatment of cn to save the extremity or delay major amputation are still being developed . foot reconstruction , resection of bony prominences , and major amputations are considered for the surgical treatment ( table 3 ) . major amputations in cn ( generally we prefer the below - the - knee amputation ) are still the current solution . if carried out properly , if the healing is complete , and if the patient is equipped in the prosthetic and rehabilitation department with a suitable prosthesis and has an adequate walking regime as part of the rehabilitation , then we know from experience that these patients , though initially perhaps unwilling to undergo surgery , are more satisfied compared to those who use orthosis for a long time , who required constant dressings of ulcerations and repeated visits to hospital . based on our personal experiences , we use transcutaneous oxygen tension more than 35 mmhg as a predictive factor for successful healing of below - knee amputation . in dialysis patients we deal with problem of a suitable prosthesis after major amputation due to changes of extremity volume between dialyses . bone resections are done as a separate intervention in isolated bone prominences , mostly in cases of a high bony pressure that can not be accommodated with orthotic and prosthetic means and in stable plantigrade foot . in some cases , a strayer procedure or achilles tendon prolongation such intervention carries a risk of instable foot in the case of larger bone resection . a bone resection is also done as preparation for reconstruction of the foot in case an infected ulceration is present or if there is suspicion of osteomyelitis . with regard to poor bone quality and the presence of neuropathy in long - term healing , the so - called superconstruction principles for reconstruction operations have been set up : ( a ) extending arthrodesis beyond the affected area on neighbouring joints , ( b ) resection of the bone for mild shortening of the foot enabling adequate repositioning of the deformity without excessive tension of soft tissues , thus helping prevent secondary ischaemisation , ( c ) usage of the strongest possible implant which can be tolerated , and ( d ) introduction of an implant that can maximally increase mechanic stability , which is the main goal . in general we can use different types of external fixators or internal fixators according to the type of deformity and preference of the surgeon . in the case of external fixators , the most suitable enabling gradual correction seems to be ankle - foot fixators of the ilizarov type or the taylor spatial frame . mostly , a three - plane fixation that combines common types of external fixators is used . an advantage of this method is the absence of internal implant that may increase the risk of infection and the possibility of earlier weight bearing on the foot . as for the internal fixations , plates are recommended if the implantation is from the plantar side , although nowadays angle stable plates are used more often . they enable good stability in an osteoporotic bone and greater variability from the point of view of plate placement . the disadvantage is the need for a wider surgical approach and problematic healing with the exposed implant . in our department when the resection is done and the reposition is finished we use them to fix intramedullary both the medial and lateral columns and subsequently apply plaster cast fixation . by using such a technique we eliminate the disadvantages of using the plates as mentioned above and we did not observe any osseous healing failure reporting by some authors [ 43 , 44 ] . most of the earlier operations have been carried out only in the chronic , inactive stage . in the active stage , an inflammatory reaction with oedema and osteoporosis are present , thus increasing the risk of complicated healing . on the other hand , this stage enables easier corrections than in the fixed deformity as it is possible to use the remodeling capacity of the bone . indications for surgery in the active stage are heavy instability , progression of the deformity , prevention of the dislocation of fragments by muscle contraction , and the general failure of conservative treatment . the position of the foot is gradually corrected by this external fixator into the correct plantigrade position , then an arthrodesis of joints is carried out , and the fixator is left in place for at least three more months . nevertheless , to date no sufficient relevant studies have been published demonstrating the success rate of the surgeries carried out during this active stage . in our department sanders and frykberg classified individual localizations of cn on the foot ( table 4 ) . in this case prevailing resorptive changes were creating deformities of the metatarsal bones of the so - called candy bar type ( figure 3 ) . popelka recommends a fixation with the use of two screws as sufficient for this surgery . on the basis of our experience we recommend using plates in cn in neuropathic terrain . in the case of heavy deformities or it is necessary to distinguish cn and osteomyelitis as mistaking cn for osteomyelitis may often lead to transmetatarsal amputation . translation of metatarsi medially or laterally is usually associated with lowering the medial column and valgus heel . a frequent consequence is abduction of the forefoot , which includes a perinavicular affection with the talus and navicular bone in plantar flection while the cuneiform bone is dislocated dorsally with the i metatarsus . the contraction of the tibialis anterior muscle worsens the deformity and practically excludes successful conservative treatment . this type of cn is very often combined with the following type iii , which is why surgical treatment of both these types will be described together . moreover , a normal position of the hindfoot is a prerequisite for the correction of the forefoot . this plantar bone prominence causes chronic ulcerations which do not respond to conservative therapy ( figure 4 ) . it is necessary to do reconstruction and stabilisation of the medial and lateral column and , in case of persistent instability , subtalar arthrodesis as well . one advantage is regional anaesthesia and minimal usage of a tourniquet . in the case of pes equinus , the first phase , according to the preoperative assessment , involves either the strayer procedure or , more frequently , prolongation of the achilles tendon , which can be carried out using either a z - plasty or a technique of three mini - incisions three cm apart from each other and up to one - half of the tendon diameter ( most frequently a lateral - medial - lateral incision ) . the position of the sole is corrected up to 90 degrees in respect to the long axis of the fibula , with the knee in full extension . this procedure restores the positive inclination of the calcaneus and facilitates the reconstruction of the medial part of the tarsus . care must be taken to avoid pes calcaneus by overextended prolongation ; this position causes heel ulcerations that do not heal , leading to the necessity for below - knee amputation . we temporarily fix the corrected position of the hindfoot with the help of the kirschner wire from the calcaneus to the tibia . in the next phase we make a slightly s - shaped incision on the medial side of the foot from the talus to the base of the i metatarsus , where we identify individual joint dislocations . earlier we used the approach to the lateral column according to ollier which led to a higher percent of secondary healing . that is why , in a deformity where the cuboid bone is the lowest bone , the callus is excised from a plantar approach . reconstruction of the lisfranc joint follows , and we correct abduction or adduction deformity performing osteotomy using the previously introduced kirschner wires . we temporarily fix the corrected position with the help of kirschner wire . in the third phase the navicular and cuboid bones are usually plantarly dislocated , with the goal being resectional talonavicular , naviculocuneiform , and calcaneocuboid arthrodesis in the corrected position , which is again temporarily fixed by the kirschner wires . a final fixation with a midfoot fusion bolt of 6.5 mm ( depuy / synthes ) is the last step . we introduce the implant medially over the head of the i metatarsus according to the deformity of the hallux from a dorsal or plantar approach up to the talus bone . laterally we introduce the implant from the mini - incision from the area of base of the iv metatarsus through the cuboid bone to the calcaneus . to date we did not have to address residual instability between the talus and calcaneus , for which subtalar arthrodesis with the same implant is recommended . we use resected bones as local autografts , reinsert tendon attachments of the medial column , and insert redon drains and the wounds are sutured . finally , we apply a padded plaster cast according to the type of intervention ( figure 5 ) . in this type the ankle and frequently a subtalar joint are most affected . because of the instability the deformity progresses and calluses and ulcerations emerge . we indicate arthrodesis of the ankle and the subtalar joint along with an external fixation ( figure 6 ) . healing complications have been observed by us in these cases . in the case of severe deformities is healed it is necessary to use an orthosis for several months ( figure 7 ) . this is the least frequent type affecting the calcaneus ( figure 8) . with regard to the poor quality of bone and thus to the retention of osteosynthetic material and the risk of infection surgical treatment is considered in the case of progressing deformity when a dislocation of fragments occurs as a result of contracting achilles tendon . the primary goal is the stability of the hindfoot and preventing the formation of ulcerations . postoperative care depends on the type of corrected deformity , the implant used ( internal or external fixation ) , the course of healing , whether or not the contralateral extremity is affected , and the ability of nonweight bearing on the operated extremity . in general a great deal of attention must be paid to the appropriate off - loading in the early and subacute postoperative stage and in the case of chronic cn in terms of localization . the advantage of external fixation is the possibility of earlier weight bearing ; internal fixation is supplemented by a plaster cast usually for three to four months . after the external fixator or plaster cast fixation is removed , individual orthoses are applied , and weight bearing is gradually increased by reduction in limitation of walking time and speed . a suitable time for using the orthosis is up to one year . after this period a lifelong follow - up including diabetes , nutrition , and infection control by antibiotic treatment if necessary is essential . thanks to new findings from the aetiopathogenesis of the condition and its biomechanics it is possible , in indicated cases , to supplement cn treatment with reconstructive procedures along with suitable implants , thus avoiding major amputation . nevertheless , to evaluate the benefits and risks of these procedures further evidence - based studies will be necessary .

myeloproliferative neoplasms ( mpns ) are a heterogeneous group of clonal diseases characterized by the excessive and chronic production of mature cells from one or several of the myeloid lineages . recent advances in the biology of mpns have greatly facilitated their molecular diagnosis since most patients present with mutation(s ) in the jak2 , mpl , or calr genes . yet the roles played by these mutations in the pathogenesis and main complications of the different subtypes of mpns are not fully elucidated . importantly , chronic inflammation has long been associated with mpn disease and some of the symptoms and complications can be linked to inflammation . moreover , the jak inhibitor clinical trials showed that the reduction of symptoms linked to inflammation was beneficial to patients even in the absence of significant decrease in the jak2-v617f mutant load . these observations suggested that part of the inflammation observed in patients with jak2-mutated mpns may not be the consequence of jak2 mutation . the aim of this paper is to review the different aspects of inflammation in mpns , the molecular mechanisms involved , the role of specific genetic defects , and the evidence that increased production of certain cytokines depends or not on mpn - associated mutations , and to discuss possible nongenetic causes of inflammation . charcot neuropathic osteoarthropathy of the foot is a relatively common complication of diabetic neuropathy . incorrect diagnosis and improper treatment often result in the extremity having to be amputated . this paper summarises the current view on the etiology , diagnostics , and treatment of diabetic charcot neuropathic osteoarthropathy , with particular focus on preserving the extremity through surgical intervention from our own experiences .

packaging is a link connecting production with marketing whereby the goods reach from the production center to the consumers in a safe and sound condition with a minimum overall cost . packaging can also be defined as the coordinated system which can enclose or protect the products for distribution , storage , preservation , transportation , information , and sales . the pharmaceutical brands are most vulnerable due to their higher market share , ease of production , and greater profit margins . products sidetracked from their proper distribution channel , or sold past their expiry date , or by modification of the package are associated with the problem of counterfeiting . counterfeits are unauthorized reproductions of a trademarked brand , which are closely similar or identical to genuine articles . the first international meeting on counterfeit medicines was held during april 13 , 1992 , at world health organization ( who ) in geneva and the following definition was accepted : a counterfeit medicine is one which is purposely and falsely mislabeled in accordance to identity and/or source . counterfeiting can pertain mutually to both branded as well as generic products comprising of either correct or incorrect ingredients , lacking active ingredients , or with forged packaging . later on , the concept was modified by the nigerian national agency for food and drug administration and control ( nafdac ) as those medicines with the same quantity of active ingredient as that of genuine brand , insufficient or no active ingredients , medicines which are post expiry date , herbal preparations that are toxic or ineffective and medicines which do not bear the name and address of the manufacturer are counterfeit . counterfeiting is a high - volume , high - profit business which causes the infringement of intellectual property rights , medicine legislations , and other aspects of criminal law . counterfeiting and piracy are in term the same since they are both the reproduction of identical copies of the genuine product . the most common counterfeit drugs in industrialized or developed countries are so - called lifestyle drugs . counterfeit drugs are the major cause of morbidity , mortality , and loss of confidence in the healthcare system . in india , the consumers reported a wider use of counterfeit and pirated products through reuse , repair , and refill of products . repackaging is one of the sources of fake drugs in europe and the united states . estimates put the total loss of life to counterfeit pharmaceuticals between 500,000 and 1,000,000 people per year . according to the bcc research report , the anticounterfeit packaging technology in the united states increased to approximately $ 34.2 billion in 2006 . sales are likely to reach $ 43 billion in 2012 , viewing a compound annual growth rate ( cagr ) of 3.2% over the tenure of 5 years from 2007 to 2012 , while as per the statement released by the organization for economic co - operation and development ( oecd ) , three - fourths of forged drugs supplied world over have some origin in india , followed by 7% and 6% from egypt and china , respectively . as of 2005 , oecd estimates the international trade of counterfeit goods to be $ 200 billion per year . china ranks first for the counterfeit products , the united states is at the second and india at the fourth rank globally . who estimates that up to 1% of medicines available in the developed world are likely to be counterfeits . this figure rises to 10% globally , although in some developing countries the estimation is about one - third for the counterfeit medicines . medicines have been the center of attraction for the counterfeiters due to the number of reasons listed in table 1 , and the factors responsible for the counterfeiting are shown in figure 1 . the medicines counterfeited worldwide with their outcomes are mentioned in the table 2.[2432 ] medicine as a target for counterfeiting drivers of counterfeiting counterfeit medicines globally authentication is of utmost importance because the use of counterfeit medicines can be harmful to the health and wellbeing of the patients . overt features are expected to assist the users to confirm the genuineness of a pack . the process can be untidy and does not always provide the print quality necessary for creating small codes , which must stay clear for weeks or months . barcodes are high - density linear or two - dimensional codes incorporated onto the product package , which are scanned and sent to the central database as shown in figure 2 . users must make sure that there is a sufficient print contrast between light and dark bars to produce a legible representation . package showing two - dimensional barcodes , scanned and sent to the central database holography is well known for its capacity to produce striking three - dimensional images , which are difficult to get through with the conventional photography . a major benefit of this process is that they can be reformed under white light . holograms are generated from the interference patterns obtained through the contact of laser beams by either angular image or laser technology . such high - definition holograms are used as a security feature on the product bottle as shown in figure 3 . the complexity of the hologram varies from the traditional three - dimensional images to computer - generated two - dimensional diffraction patterns . holograms are now widely available in variety of formats such as holographic shrink sleeves , blister packaging aluminum foil , holographic induction cap seals , polyester - based tamper evident labels , and holographic hot stamping foil . but still it is reported that more than half the sales of the artesunate drug in south east asia is forged , despite the presence of the hologram.[4253739 ] the overall advantages and disadvantages of overt technologies are described in table 3 . bottle with a hologram as a security feature advantages and disadvantages of overt and covert technologies the rationale of a covert feature is to aid the brand owner to recognize a counterfeited product . the general public will not be aware of its presence nor will have the resources to confirm it . a covert feature should be difficult to sense or copy without the specialist knowledge . the highly appreciated though expensive technology used presently is the radio frequency identification device ( rfid ) . , rfid includes batch information which can be interrogated at a distance without requiring the line of sight . the rfid allows the recognition of objects through wireless communications in a set frequency band . three vital components in any rfid system are the tag , the reader , and the software . the tag is an integrated circuit containing an exclusive tracking verifier , called an electronic product code ( epc ) , which is transmitted via electromagnetic waves in the radio spectrum . the reader captures the transmitted signal and provides the network connectivity . for their track and trace usage , the diverse rfid tags that are used . the high cost of a rfid tag ( 2050 cents as opposed to a 2 cent barcode ) might forbid the companies from implementing it . the biometric fingerprints introduced by the bayer group utilize 1-mw diode lasers to analyze the innate surface makeup of each item . microscopic irregularities which are caused by the setting of paper fibers or plastic result in spreading of the laser beam by a laser speckle event . by measuring the dispersal of the light at various angles , surface uniqueness is recorded . overt features are expected to assist the users to confirm the genuineness of a pack . the process can be untidy and does not always provide the print quality necessary for creating small codes , which must stay clear for weeks or months . barcodes are high - density linear or two - dimensional codes incorporated onto the product package , which are scanned and sent to the central database as shown in figure 2 . users must make sure that there is a sufficient print contrast between light and dark bars to produce a legible representation . package showing two - dimensional barcodes , scanned and sent to the central database holography is well known for its capacity to produce striking three - dimensional images , which are difficult to get through with the conventional photography . a major benefit of this process is that they can be reformed under white light . holograms are generated from the interference patterns obtained through the contact of laser beams by either angular image or laser technology . such high - definition holograms are used as a security feature on the product bottle as shown in figure 3 . the complexity of the hologram varies from the traditional three - dimensional images to computer - generated two - dimensional diffraction patterns . holograms are now widely available in variety of formats such as holographic shrink sleeves , blister packaging aluminum foil , holographic induction cap seals , polyester - based tamper evident labels , and holographic hot stamping foil . but still it is reported that more than half the sales of the artesunate drug in south east asia is forged , despite the presence of the hologram.[4253739 ] the overall advantages and disadvantages of overt technologies are described in table 3 . bottle with a hologram as a security feature advantages and disadvantages of overt and covert technologies the rationale of a covert feature is to aid the brand owner to recognize a counterfeited product . the general public will not be aware of its presence nor will have the resources to confirm it . a covert feature should be difficult to sense or copy without the specialist knowledge . the highly appreciated though expensive technology used presently is the radio frequency identification device ( rfid ) . , rfid includes batch information which can be interrogated at a distance without requiring the line of sight . the rfid allows the recognition of objects through wireless communications in a set frequency band . three vital components in any rfid system are the tag , the reader , and the software . the tag is an integrated circuit containing an exclusive tracking verifier , called an electronic product code ( epc ) , which is transmitted via electromagnetic waves in the radio spectrum . the reader captures the transmitted signal and provides the network connectivity . for their track and trace usage , the diverse rfid tags that are used the high cost of a rfid tag ( 2050 cents as opposed to a 2 cent barcode ) might forbid the companies from implementing it . the biometric fingerprints introduced by the bayer group utilize 1-mw diode lasers to analyze the innate surface makeup of each item . microscopic irregularities which are caused by the setting of paper fibers or plastic result in spreading of the laser beam by a laser speckle event . by measuring the dispersal of the light at various angles , surface uniqueness is recorded . authentication in packaging utilizes mainly the concept of sealing which has been significantly demonstrated by american bank note holographics . holoseal is a patented pressure - sensitive , tamper apparent holographic security label that features a customized tamper apparent fracture pattern , black light verification system , and machine - readable entrenched code . for the tracking purpose , holoseal can be numbered or personalized to give an account of region , plant , or product . holocap comprises several different heat - sealable films which are attached directly to the containers using the induction seal technology . these different films unite to form a single holographic inner seal that provides a higher level of defense to containers or bottles . package sealing tapes the trend of utilizing sealing in packaging for the development of anticounterfeiting techniques changes with the upcoming concept of lasers and forensic approaches . holospot is a discrete forgery - proof information carrier that can be attached to any product . it uses laser encryption of computer - generated lithograms into small polymeric data carriers and offers multiple overt and covert security features , whereas on the other hand ident seal is a visible text or high - contrast barcode inscribed by lasers . the anticounterfeit label forge guard of fujifilm corporation can visualize full color images or text clearly with a special viewer . this label regulates light wavelengths on the nano - optic level , so those with the viewer can see hidden full color images . still the need was for the invisible substrates to enhance the security and the research led to the development of forensic markers . these forensic markers include physical , chemical , and biological taggants . a merck product , esan features numerous hidden and forensic security features on the pack from its securalic product line . the security features are built into the ivy leaf design element printed on the folded box . other systems include a tamper evident packaging system , film wrappers , and breakable caps . the tamper evident feature helps to maintain the integrity of the drug product by preventing the profit of counterfeiters by either repacking or reselling of the pharmaceutical products as shown in figure 5 . a film wrapper is a transparent film with a characteristic design covered securely around a product or product container . the film must be cut or torn to open the container and remove the product . substrate options include ultradestructible films , solvent - sensitive papers , and voidable films that provide images when removed as shown in figure 6 . breakable caps , as the name suggests , break when an effort to open is made . these caps provide external tamper evidence and can also be combined with the internal seals , thereby assisting with double security . the overall choice of anticounterfeit techniques at various levels of packaging has been described in table 4 . external and internal tamper evident feature label that leaves a void mark on tampering choice of anticounterfeit solutions at different packaging levels medicines save lives and prevent diseases and epidemics only if they are safe , efficacious , of good quality , and are rationally used . the use of unsafe , substandard , ineffective , and counterfeit medicines can be harmful to the health and wellbeing of the patients . governments must establish the national medicine regulatory authority ( nmra ) to monitor the quality of medicines in the market to detect and prevent any substandard and counterfeit medicines from reaching the public , by working closely with national law enforcement agencies such as the police and custom officers . the nmra must ensure that all medicine manufacturing , import , export , and distribution activities are carried out in premises approved by the nmra , and that individuals and companies engaged must have licenses to operate such activities . the counterfeit pharmaceuticals initiative ( cpi ) was launched by the icc in 2003 for the creation of a counterfeit pharmaceutical database with online search facility;construction of a dedicated cpi website;liaising with regulators;providing assistance to members by lobbying and investigation;special projects and surveys , e.g. , internet pharmacies ; andthe implementation of anticounterfeiting technologies . the creation of a counterfeit pharmaceutical database with online search facility ; construction of a dedicated cpi website ; liaising with regulators ; providing assistance to members by lobbying and investigation ; special projects and surveys , e.g. , internet pharmacies ; and the implementation of anticounterfeiting technologies . the implementation of overt and covert techniques by the industries can raise the bar for the counterfeiters , but the final awareness must be at the consumer level . the consumers should buy medicines only from licensed pharmacies and medicine outlets , be suspicious of heavily discounted medicines , and check if the packaging indicates the batch number , manufacturing date , expiry date , and the manufacturer 's name . counterfeit drugs not only affect the sick and innocent consumers but also the general public , and deserve more attention . chemotherapeutic drugs , alone or as an adjuvant therapy to surgery and radiation , are a vital part in cancer treatment . however , their effectiveness is often offset by severe side effects caused by poor selectivity and toxicity to normal cells . in recent years , the rapid advance in the fields of bioinformatics and genomics facilitated the identification of numerous target molecules that are uniquely or overly expressed in cancer cells , including certain receptors and enzymes . these discoveries were successfully utilized for the specific delivery of chemotherapeutic drugs to tumor cells , with the double goal of increasing their therapeutic efficacy while decreasing their toxicity to normal cells . the enzyme dipeptidyl peptidase iv ( dppiv ) ( ec 3.4.14.5 ) , also known as cd26 ( t - cell surface activation antigen ) or adenosine deaminase ( ada ) , was first identified in 1966 . dppiv is a member of the prolyl oligopeptidase ( pop ) family of serine proteases and specifically hydrolyzes the peptide bond c - terminal to proline or alanine in the penultimate p1 position . it is normally present on the apical membrane surface of several cell types , including t cells and b cells , natural killer ( nk ) cells , epithelial cells of kidney , intestine brush border membranes , plasma , and endothelial cells of blood vessels , and is involved in many diverse physiological processes . in addition , it was recently discovered that dppiv is overly expressed in human renal cell carcinoma tissues , and it is thought to play a role in the pathogenesis of other various human cancers as well . moreover , proline - containing substrates are hydrolyzed exclusively by proline - specific enzymes and are less susceptible to nonspecific peptidases and proteases . thus , a dppiv - cleavable prodrug containing a proline dipeptide conjugated to a cytotoxic chemotherapeutic agent may limit nonspecific activation . the purpose of this study was to investigate the feasibility of exploiting dppiv as a prodrug activating enzyme , to allow specific targeting of chemotherapeutic agents to cancer cells . to that end , the expression profiles of dppiv in 60 cancer cell lines ( nci 60 ) were obtained , and 7 cell lines , which represent the spectrum of dppiv expression , were selected . a candidate prodrug was then synthesized by linking the cytotoxic drug melphalan ( figure 1a ) to a proline dipeptide , creating glycyl - prolyl - melphalan prodrug ( gp - mel ; figure 1b ) with expected dppiv affinity . functional activity studies of dppiv with the prodrug , in the absence vs presence of inhibitor , confirmed that the prodrug is a specific dppiv substrate . finally , hydrolysis and cell proliferation studies were performed in high and low dppiv expression cancer cell lines . overall , this work indicates that dppiv may be exploited as a prodrug activating enzyme for efficient chemotherapeutic drug targeting . chemical structures of ( a ) melphalan and ( b ) its gly - pro dipeptide prodrug , gp - mel . melphalan , porcine kidney dipeptidyl peptidase ( porcine dppiv ) , glycyl - prolyl - p - nitroanilide ( gp - pna ) , glycyl - phenylalanyl - p - nitroanilide ( gf - pna ) , glycyl - arginyl - p - nitroanilide ( gr - pna ) , p - nitroaniline ( pna ) , diprotin a ( ile - pro - ile ) , xtt , pms ( n - methyldibenzopyrazine methyl sulfate ) , and ladder sybr green were purchased from sigma chemical co. ( st . louis , mo ) . n , n - dimethylformamide ( dmf ) , piperidine , triisopropylsilane ( tis ) , triethanolamine ( tea ) , and trifluoroacetic acid ( tfa ) were purchased from aldrich chemical co. ( milwaukee , wi ) . boc - l - gly - l - pro , 1-hydroxybenzotriazole anhydrous ( hobt ) , 2-(1h - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) , triisopropylsilane ( tis ) , n-(9-fluorenylmethoxycarbonyloxy)succinimide ( fmoc - osu ) , and wang resin ( 100200 mesh ) were obtained from calbiochem - novabiochem ( san diego , ca ) . access rt - pcr kit , dna blue / orange loading dye , 1 kb ladder and 100 bp ladder were obtained from promega ( madison , wi ) . 420% tbe gels , trizol reagent , and custom - ordered dppiv sense and antisense primers were from invitrogen life technologies ( carlsbad , ca ) . the cancer cell lines igrovi ( ovarian ) , pc-3 ( prostate ) , 786 - 0 ( renal ) , sk - mel-5 ( melanoma ) , sk - ov-3 ( ovarian ) , caco-2 ( colon ) , and hepg2 ( liver ) were obtained from national cancer institute or atcc . dulbecco s modified eagle s medium ( dmem ) , rpmi-1640 , fetal bovine serum ( fbs ) , fetal calf serum ( fcs ) , phosphate buffered saline ( pbs ) , and trypsin - edta were purchased from gibco brl ( grand island , ny ) . minimal essential medium ( mem ) was obtained from atcc . all hplc grade solvents ( acetonitrile , dmf , ethyl acetate , and piperidine ) used for peptide synthesis or hplc analysis trifluoroacetic acid for hplc analysis was obtained from pierce ( rockville , il ) . the solid - phase synthesis of the l - glycyl - l - prolyl prodrug of melphalan was carried out in a stepwise fashion with wang resin as described below and is summarized in the schematic shown in figure 2 . fmoc - l - melphalan synthesis : fmoc - osu ( 300 mg , 0.89 mmol ) was added to an ice - cold solution of melphalan , 1 ( 200 mg , 0.82 mmol ) , in a mixture of acetone ( 20 ml ) , distilled water ( 10 ml ) , and nahco3 ( 300 mg ) . the mixture was stirred for 1 h at 0 c and then reacted at room temperature for 16 h. after the reaction was complete , the mixture was concentrated . 15 ml of ethyl acetate and 15 ml of distilled water were added . the aqueous phase was then extracted with ethyl acetate ( 3 15 ml ) . the combined organic phases were washed with distilled water and brine and dried over mgso4 . the filtrate was concentrated under vacuum to yield fmoc - l - melphalan , 2 ( 330 mg , yield 98% ) . fmoc - l - melphalan ( 330 mg ) and wang resin ( 0.1 mmol ) were added into a 100 ml round reaction bottle with 20 ml of dmf . hobt ( 0.3 mmol ) , hbtu ( 0.3 mmol ) , and tea ( 0.3 mmol ) were then added to the resin mixture and stirred overnight at room temperature . the wang resin , 3 , was washed 3 times with 10 ml of dmf after filtration and then washed 3 times for 10 min each in 10 ml of 20% piperidine in dmf . boc - l - glycyl - l - proline ( 0.3 mmol ) and wang resin 3 were added to 20 ml of dmf in a 100 ml round reaction bottle . hobt ( 0.3 mmol ) , hbtu ( 0.3 mmol ) , and tea ( 0.3 mmol ) were added and stirred overnight at room temperature . the resultant wang resin , 4 , was washed 3 times with 10 ml of dmf after filtration and then washed 3 times for 10 min each in 10 ml of 20% piperidine in dmf . wang resin 4 was then added to 10 ml of cleavage buffer ( tfa : tis : h2o , 95:2.5:2.5 ) and stirred for 1 h at room temperature . after filtration , the solution was concentrated and cold ether ( 10 ml ) was added to precipitate out the prodrug 5 . after removing ether , the residues were reconstituted with 5 ml of distilled water and lyophilized . the purity of the melphalan prodrug was determined by hplc to be 95% . electrospray ionization mass spectra ( esi - ms ) were obtained on a thermoquest lcq electrospray ionization mass spectrometer . the observed molecular weight of the prodrug was found to be consistent with that required by its structure . the structural identity of the prodrug was then confirmed using proton nuclear magnetic resonance spectra ( h nmr ) . schematic of solid - phase synthesis of l - glycyl - l - prolyl - melphalan prodrug . l - glycyl - l - prolyl - melphalan : yield 13% ; percent purity 95% ; h nmr ( dmso - d6 ) 1.952.20 ( m , 4h , ch2 on proline ) , 3.053.35 ( m , 12h , ch2 ) , 4.444.80 ( m , 5h , ch , ch2 ) , 6.506.90 ( m , 4h , phenol h ) ; esi - ms 459.4 ( m + h ) . the microarray data and the u95av2 affymetrix data were downloaded from the dtp database ( https://wiki.nci.nih.gov/display/ncidtpdata/molecular+target+data ) . the programming language perl was used to sift through more than 26,000 genes to find all enzymes with names that end with the suffix ase . the enzyme text file was then used to separate the enzymes into different classes , such as hydrolases , peptidases , and esterases as described earlier . visual basic was then used to arrange the data retrieved so that the expression of each enzyme gene in the 60 cancer cell lines could be easily visualized using visual tools such as cluster and treeview programs . the arranged data from peptidases was normalized using a pool of 12 cell lines ( nci - h226 , colo 205 , mcf-7 , hs 578 t , ovcar-3 , ovcar-4 , k-562 , hl-60 ( tb ) , caki-1 , lox imvi , pc-3 , snb-19 ) as reference and clustered according to the origin of the cell lines . the sorted expression data for the 60 cancer cell lines was clustered using hierarchical clustering , and a file was created ( .cdt ) such that the expression of the genes in 60 cancer cell lines could be visualized using treeview . the potential enzyme target , dppiv , was selected on the basis of the differential expression in various cell lines or tissues with emphasis on high expression in a particular tissue as compared to other tissues and high substrate specificity of the enzyme . candidate cancer cell lines were selected from the nci 60 cell lines based on the expression levels of dppiv , doubling time , and growth requirements . thus , the cancer cell lines selected , igrovi , pc-3 , 786 - 0 , sk - ov-3 , and sk - mel-5 , represent cells with high , medium , and low expression of dppiv , reasonably short doubling times , and standard growth requirements . in addition , caco-2 as well as hepg2 cell lines were also used in the studies . total rna was lysed and purified from each cancer cell line with trizol reagent according to the manufacturer s protocol . isolated rna ( 0.5 g ) was reverse transcribed to cdna with amv reverse transcriptase , oligo ( dt ) , and access rt - pcr kit following the standard protocol . each of the 20 cycles of amplification consisted of denaturation ( 94 c for 25 s ) , annealing ( 50 c for 25 s ) , and extension ( 68 c for 40 s ) . the primer set used for dppiv amplification was the sense primer ( 5-ccttctactctgatgagtcactgc-3 ) and the antisense primer ( 5-gtgccactaagcagttccatcttc-3 ) . pcr product was identified by electrophoresis in 420% tbe gels followed by sybr green staining . the gel was then visualized with uv light , and the relative intensities of the rt - pcr product bands were measured using metamorph software . porcine kidney dppiv was used in the studies due to its commercial availability and its close alignment with human dppiv ( 88% homology ) . stock solutions of porcine dppiv were prepared by reconstituting lyophilized powder ( 0.87 unit , 23 units / mg ) in 1 ml of 0.1 m tris - hcl ( ph 8.0 ) and stored at 80 c . solutions of the various substrates glycyl - prolyl - p - nitroanilide ( gp - pna ) , glycyl - phenylalaninyl - p - nitroanilide ( gf - pna ) , or glycyl - argininyl - p - nitroanilide ( gr - pna ) at 0.4 mm were mixed with appropriate amounts of diluted dppiv solution ( final dppiv concentration in mixture , 0.473 g / ml ) and incubated at 37 c for 1530 min in flat - bottom 96-well plates . the hydrolysis of the substrates was monitored by measurement of absorbance at 405 nm ( generation of p - nitroanilide ) using a precision microplate reader ( emax , molecular devices ) . the competitive inhibition of hydrolysis of the substrates by porcine kidney dppiv in the presence of the inhibitor diprotin a was determined by including 0.4 mm diprotin a in the diluted dppiv mixture prior to incubation . initial hydrolysis rates were determined in all hydrolysis experiments by assaying the amounts of pna released . dppiv activity in the selected cancer cells was determined using the standard substrate gp - pna . briefly , cells were grown to 90% confluence in 150 mm culture plates , washed carefully with 0.15 m nacl , and collected by scraping . the cells were resuspended in 0.15 m nacl and then centrifuged at 3,000 rpm for 5 min . the cell pellet was resuspended in phosphate buffered saline ( pbs , ph 7.4 ) , and sonicated for 5 s two times on ice . the sonicated cell suspension was centrifuged at 18000 g for 30 min at 4 c . the supernatant was then used in hydrolysis studies , and to determine protein content ( bio - rad dc protein assay ) . the protein content was adjusted to approximately 1000 g / ml by appropriate dilutions before use in hydrolysis studies . hydrolysis studies were conducted in a 96-well microplate containing the cell homogenate suspensions at 37 c . the reactions were initiated by the addition of gp - pna ( final concentration 1 mm ) to the suspensions . the absorbance was monitored every minute for a total of 3060 min using a precision microplate reader ( emax , molecular devices ) at 405 nm . in the competitive inhibition studies , diprotin a at 1 mm was included along with 1 mm gp - pna , and the mixture was added to the cell homogenate suspensions and absorbance monitored as described above . dppiv activity was expressed as the amount of pna ( micromoles ) released per minute , normalized to the amount of protein . hydrolysis of the melphalan prodrug by porcine kidney dppiv and the effect of the competitive inhibitor diprotin a were assayed using 96-well microplates . porcine dppiv enzyme solution ( 230 l at a final concentration 4.4 g / ml ) was placed in triplicate wells maintained at 37 c . the reactions were initiated by the addition of gp - mel ( 1 mm final concentration in mixture ) , and 40 l aliquots were removed at predetermined time points and added to two volumes of 10% ice - cold tfa to quench the reaction and precipitate protein . in the inhibition studies , diprotin a and gp - mel were both added ( 1 mm final concentration ) to the enzyme solution , incubated at 37 c , and sampled as described above . the quenched precipitated samples were then filtered through a 0.45 m filter plate and centrifuged at 1800 g and 4 c for 20 min . the extent of hydrolysis of gp - mel in caco-2 and sk - mel-5 cell homogenates was determined as follows . caco-2 and sk - mel-5 cells and cell homogenates were prepared as described earlier . the hydrolysis reactions were carried out in 96-well plates ( corning , corning , ny ) . 230 l of the cell suspensions ( 1000 g / ml protein ) were placed in triplicate wells and the reactions initiated by the addition of gp - mel ( final concentration 1 mm in mixture ) and incubated at 37 c . at predetermined time points 40 l aliquots were removed and added to two volumes of 10% ice - cold tfa to quench the reaction and precipitate protein . in the inhibition studies , diprotin a and gp - mel were both added ( 1 mm final concentration ) to the cell suspensions , incubated at 37 c , and sampled as described above . the quenched precipitated samples were then filtered through a 0.45 m filter plate and centrifuged at 1800 g and 4 c for 20 min . dppiv activity was expressed as the amount ( picomoles ) of melphalan released per minute , normalized to the amount of protein . the concentrations of gp - mel and melphalan were determined on a waters hplc system ( waters inc . , the hplc system consisted of two waters pumps ( model 515 ) , a waters autosampler ( wisp model 712 ) , and a waters uv detector ( 996 photodiode array detector ) . the system was controlled by waters millennium 32 software ( version 3.0.1 ) . samples were injected onto a waters xterra c18 reversed phase column ( 5 m , 4.6 250 mm ) equipped with a guard column . the flow rate was 1 ml / min , and the mobile phase was 70:30 ( % standard curves generated for prodrug and parent drug were utilized for quantitation of integrated area under peaks . cell proliferation assays were conducted to determine the cytotoxic activities of the prodrug gp - mel and the parent melphalan . the assays were carried out with caco-2 and sk - mel-5 cells since the expression of dppiv was found to be highest and lowest , respectively , in these cells , based on rt - pcr expression results . caco-2 and skmel-5 cells were plated overnight in a 96-well cell culture plate at a density of 5,000 cells / well per 0.1 ml . stock solutions ( 1 mm ) of gp - mel and melphalan were prepared in rpmi-1640 phenol red free medium supplemented with fbs . stock solutions were serially diluted to obtain a total of six drug concentrations , 1 mm , 0.5 mm , 0.25 mm , 0.125 mm , 0.0625 mm , and 0.03125 mm , for cell proliferation studies . after 24 h , the medium in the 96-well plate was aspirated and replaced with drug solutions in the medium . the cells were then incubated at 37 c and 5% co2 for 48 h. after 48 h , 50 l of xtt labeling mixture ( 5 ml of 1 mg / ml xtt in rpmi-1640 phenol red free medium mixed with 100 l of 0.383 mg / ml pms in phosphate buffered saline ) was added to each well . the color development , due to formation of formazan dye by metabolically active cells , was monitored for 4 h , after which the plates were read at 490 nm ( 805 nm as the reference wavelength ) with a precision microplate reader ( emax , molecular devices ) . the percent cell viability , at different drug and prodrug concentrations , relative to control was then plotted as a function of drug / prodrug concentration to compute the gi50 values . after the genes had been sorted as hydrolases , peptidases , and esterases , they were clustered and their expression in the 60 cancer cell lines was visualized as described previously . the expression patterns of proline - specific peptidases such as prolinases / peptidase , prolyl carboxypeptidase , prolyl oligopeptidase / prolyl endopeptidase , dppiv / cd26 , aminopeptidase p , and prolidase / peptidase d , were selected for further assessment . dppiv was overexpressed in most renal carcinoma cell lines ( uo-31 , tk-10 , sn12c , rxf 393 , caki-1 , achn , a498 , and 786 - 0 ) compared to cancer cell lines derived from other tissues . the relatively higher dppiv expression in 786 - 0 renal carcinoma cell line compared to its expression in sk - ov-3 ovarian carcinoma or sk - mel-5 melanoma cell lines was consistent with affymetrix expression data . thus , in addition to the selection of igrovi ( ovarian cancer cell line ) , pc-3 ( prostate cancer cell line ) , 786 - 0 ( renal cancer cell line ) , sk - ov-3 ( ovarian cancer cell line ) , and sk - mel-5 ( melanoma ) based on the u95av2 affymetrix gene expression of dppiv ( table 1 ) , hepg2 and caco-2 cells were also selected for characterization of dppiv activity with standard substrates and rt - pcr determinations of dppiv expression . rt - pcr of extracellular mrna in the seven selected human cancer cell lines was performed with primers specific for dppiv . the expression profiles of dppiv in the seven cancer cell lines evidenced by the band at 315 bp are shown in figure 3 . the results of semiquantitative rt - pcr analysis ( table 1 ) indicated that dppiv was expressed at high levels in caco-2 and hepg2 cells , and at very low levels in sk - mel-5 cells . thus , the relative rt - pcr expression ( expression in sk - mel-5 set at unity ) in the seven cell lines listed in table 1 were caco-2 ( 13.49 ) , hepg2 ( 12.71 ) , igrovi ( 7.92 ) , pc-3 ( 5.51 ) , 786 - 0 ( 2.81 ) , sk - ov-3 ( 1.89 ) , and sk - mel-5 cells ( 1.0 ) . the relative rt - pcr expression in the cancer cell lines exhibited an excellent linear correlation ( r = 0.94 ) with u95av2 affymetrix microarray data that was available for five of the seven cell lines ( figure 4 ) . expression profiles of dppiv ( 315 bp product ) in 7 cancer cell lines determined by semiquantitative rt - pcr : 100 base pair dna ladder ( lane 1 ) , caco-2 ( lane 2 ) , hepg2 ( lane 3 ) , igrovi ( lane 4 ) , pc-3 ( lane 5 ) , 786 - 0 ( lane 6 ) , sk - ov-3 ( lane 7 ) , and sk - mel-5 ( lane 8) . linear correlation of affymetrix genechip expression of dppiv with dppiv expression determined using rt - pcr in 5 cancer cell lines . the specific activity of a standard substrate gp - pna for porcine dppiv was determined by the release of p - nitroanilide and the resultant effect on the clarity of the solution . the baseline activity was 14.02 0.90 nmol / min/g dppiv . in the presence of the specific inhibitor diprotin a , it decreased substantially to 0.15 nmol / min/g dppiv . the kinetic parameters km and vmax obtained from a plot of v0 versus [ s ] ( figure 5 ) were 231 33 m and 0.650 0.037 mol / min / mu dppiv , respectively ( fit estimate sd ; n = 3 ) . the kcat value determined from vmax and dppiv concentration ( e0 ) was 36 s. in contrast , gf - pna or gr - pna produced no detectable release of p - nitroanilide ( negligible absorbance at 405 nm ) , indicating that these two molecules were resistant to cleavage by porcine dppiv . gp - pna hydrolysis kinetic profile with porcine dppiv ( mean sd , n = 3 ) . the activity of dppiv in various cancer cell homogenates was assessed using the standard substrate gp - pna . the results shown in figure 6 indicate that dppiv activity was in the order hepg2 caco-2 igrovi pc-3 786 - 0 sk - ov-3 sk - mel-5 cells . caco-2 and hepg2 cells exhibited high dppiv activity ( around 20 pmol / min/g protein ) whereas sk - mel-5 cells showed the lowest activity ( 1.3 pmol / min/g protein ) among the cells tested ( figure 6 ) . these results are in excellent correlation ( r = 0.96 ) with the relative rt - pcr expression of dppiv in the cell lines ( figure 7 ) . the hydrolysis of gp - pna in the presence of diprotin a was significantly inhibited in all cancer cell homogenates examined ( figure 6 ) , with an average inhibition of 95% ( range : 85100% ) . specific activity of dppiv against gp - pna ( pmol / min/g protein ) in 7 cell homogenates , in the absence vs presence of diprotin a ( mean sd , n = 3 ) . linear correlation of specific activity of dppiv against gp - pna ( pmol / min/g protein ) in 7 cancer cell homogenates with dppiv expression determined using rt - pcr . the specific activity of pure porcine enzyme against gp - mel was 1.2 0.1 nmol / min/g protein . the activity was substantially lowered to 100 pmol / min/g protein , a 93% inhibition , in the presence of diprotin a. dppiv activity against the prodrug determined in caco-2 and sk - mel-5 homogenates was 5.14 0.01 and 0.68 0.03 pmol / min/g protein , respectively , which was significantly inhibited by an average of 85% in the presence of diprotin a ( figure 8) . specific activity of dppiv against gp - mel ( pmol / min/g protein ) in caco-2 and sk - mel-5 cell homogenates , in the absence vs presence of diprotin a ( mean sd , n = 3 ) . the antiproliferative activity of gp - mel was determined in caco-2 and sk - mel-5 cancer cells and compared with that obtained with the parent drug melphalan . the percent cell viability profiles as a function of drug or prodrug concentration are shown in figure 9 . the profiles indicate that the antiproliferative action of melphalan was similar in caco-2 and sk - mel-5 cells with gi50 values of 34.9 m and 56.6 m , respectively . the gi50 value for gp - mel in caco-2 cells ( 261.3 m ) , however , was significantly lower than that obtained with sk - mel-5 cells ( 806.7 m ) . the cytotoxic activity of gp - mel in caco-2 and sk - mel-5 cells was consistent with the expression levels of dppiv in these cells . cell proliferation assay of melphalan and gp - mel after 48 h incubation with caco-2 and sk - mel-5 cells . data are expressed as mean sd ; n = 3 for each treatment at each concentration . in the past three decades , the science of molecular biology was revolutionized by the rapid advancement of the complementing fields of genomics and bioinformatics . recently , a number of enzymes from the peptidase / protease class were found to be highly expressed in several types of tumors , and to play an important role in the pathophysiology of tumor cells . dppiv has been previously associated with the onset and progression of several cancer types ; inamoto et al . demonstrated that the blockage of ddpiv reduced several cancer - related processes in the human renal carcinoma cell line caki-2 . it also reduced the tumor size and increased the survival of mice in a xenograft model . furthermore , in patients with clear cell renal cell carcinoma ( ccrcc ) , higher dppiv activity was linked with a significant decrease in patients 5 year survival rates . dppiv was also shown to be overexpressed in several human colon cancer tissues and in human colon cancer cell lines , and its inhibition reduced carcinogenesis in a rat model . in the prostate , dppiv activity in cancerous versus benign prostatic hyperplasia was increased 2-fold . an elevation of dppiv activity was also found in the prostatic secretions and the peripheral zone of the prostate , where most prostate cancers arise . higher dppiv levels in cancerous versus normal prostate tissue was correlated with psa level , tumor residue , cancer stage , and tumor size in humans . in addition to its expression and involvement in cancer , dppiv is one of the few proline - specific proteases that is able to cleave proline - associated peptide bonds , as the unique cyclic structure of proline serves as a structure regulation element which limits the susceptibility for nonspecific enzymatic degradation . thus , a high level of expression in cancer cells , combined with high substrate specificity , indicated that dppiv may be a potential target molecule for the delivery of chemotherapeutic drugs , and sparked our interest in developing a dppiv - cleavable anticancer prodrug . the functional activity of pure porcine dppiv was assessed against three gly - x dipeptide chromogenic compounds , gp - pna , a well - known standard dppiv substrate , as well as gf - pna and gr - pna , two dipeptide analogues that do not contain proline in the p1 position . the kinetic parameters obtained in this study were consistent with those previously reported for gp - pna with porcine dppiv . the negligible activity observed with gf - pna and gr - pna is consistent with dppiv substrate requirements , and eliminates the possibility of nonspecific degradation . similarly , in the seven investigated cancer cell lines , gp - pna exhibited an excellent linear correlation ( r = 0.96 ) with rt - pcr dppiv expression ( figure 7 ) , and dppiv activity was significantly inhibited in the presence of the specific inhibitor diprotin a ( average inhibition 95% ) . the similarity of the extent of inhibition in cell homogenates with that observed with porcine dppiv underscores the similarity of human and porcine dppiv . melphalan , phenylalanine mustard ( l - pam ) , is an established anticancer agent that was originally approved for the treatment of multiple myeloma . recently it has been suggested as a therapeutic agent in the treatment of a variety of cancers such as ovarian cancer , breast cancer , colorectal cancer , and melanoma . however , its use has been limited due to severe side effects such as bone marrow suppression , leukopenia , and thrombocytopenia , largely due to lack of selectivity for cancer cells , and so it was selected as the model drug for investigating our approach . a proline dipeptide prodrug of melphalan was designed to follow dppiv substrate specificity requirements . additionally , the peptide bond between p1 and p1 requires a trans - configuration . the release of melphalan from the gp - mel prodrug by pure porcine dppiv was extensive , and was 93% reduced in the presence of diprotin a , indicating that the activation of gp - mel was dppiv - dependent . this was further corroborated in caco-2 and sk - mel-5 homogenates when the prodrug activation was significantly inhibited by diprotin a ( figure 8) . the 8-fold greater activation of the prodrug in caco-2 compared to sk - mel-5 homogenates ( figure 8) was consistent with the 14-fold higher rt - pcr dppiv expression levels , and the 15-fold higher dppiv activity against gp - pna in caco-2 cells compared to sk - mel-5 cells ( figure 7 ) . as a preliminary assessment of gp - mel potential to serve as an anticancer prodrug , we have investigated the antiproliferative activity of gp - mel in caco-2 and sk - mel-5 cell lines . the antiproliferative activity of the prodrug was shown to be dependent on dppiv expression level in the cells ; the cytotoxicity ( represented by gi50 ) of gp - mel in caco-2 cells was 3-fold higher ( 260 m ) than that in sk - mel-5 cells ( 800 m ) ( figure 9 ) . in contrast , for the free parent drug melphalan , similar gi50 values were obtained in caco-2 and in sk - mel-5 cells ( 35 and 44 m , respectively ) , demonstrating the nonselective cytotoxic action of melphalan ( figure 9 ) . the cytotoxic effect of gp - mel on both cell lines was significantly lower in comparison to melphalan ; the prodrug failed to show significant cytotoxic effect in concentrations equimolar to those required for maximal growth inhibition by melphalan . this result may indicate that the gp - mel prodrug is not likely to be cytotoxic by itself , and further validates the key role of dppiv in the activation of the gp - mel prodrug . on the other hand , it may indicate insufficient activation , resulting in too low free drug levels . overall , our results demonstrate the potential to exploit dppiv as a prodrug activating enzyme for efficient chemotherapeutic drug targeting . in conclusion , dppiv was identified as a potential prodrug target due to its differential expression levels in tumor and normal tissues and relatively strict substrate specificity . a gly - pro dipeptide prodrug of melphalan , gp - mel , was designed and synthesized , based on the highly specific substrate requirements of dppiv . the finding that the activation and antiproliferative activity of gp - mel in cells were highly dependent on dppiv expression levels confirmed our hypothesis that dppiv is a feasible functional prodrug target for effective and selective chemotherapeutic action .

packaging is the coordinated system that encloses and protects the dosage form . counterfeit drugs are the major cause of morbidity , mortality , and failure of public interest in the healthcare system . high price and well - known brands make the pharma market most vulnerable , which accounts for top priority cardiovascular , obesity , and antihyperlipidemic drugs and drugs like sildenafil . packaging includes overt and covert technologies like barcodes , holograms , sealing tapes , and radio frequency identification devices to preserve the integrity of the pharmaceutical product . but till date all the available techniques are synthetic and although provide considerable protection against counterfeiting , have certain limitations which can be overcome by the application of natural approaches and utilization of the principles of nanotechnology . the efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells . recently , the enzyme dipeptidyl peptidase iv ( dppiv ) was considered as a potential target for the delivery of chemotherapeutic drugs . the purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to dppiv as a strategy to enhance their specificity . the expression profile of dppiv was obtained for seven cancer cell lines using dna microarray data from the dtp database , and was validated by rt - pcr . a prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline - glycine dipeptide moiety , followed by hydrolysis studies in the seven cell lines with a standard substrate , as well as the glycyl - prolyl - melphalan ( gp - mel ) . lastly , cell proliferation studies were carried out to demonstrate enzyme - dependent activation of the candidate prodrug . the relative rt - pcr expression levels of dppiv in the cancer cell lines exhibited linear correlation with u95av2 affymetrix data ( r2 = 0.94 ) , and with specific activity of a standard substrate , glycine - proline - p - nitroanilide ( r2 = 0.96 ) . the significantly higher antiproliferative activity of gp - mel in caco-2 cells ( gi50 = 261 m ) compared to that in sk - mel-5 cells ( gi50 = 807 m ) was consistent with the 9-fold higher specific activity of the prodrug in caco-2 cells ( 5.14 pmol / min/g protein ) compared to sk - mel-5 cells ( 0.68 pmol / min/g protein ) and with dppiv expression levels in these cells . our results demonstrate the great potential to exploit dppiv as a prodrug activating enzyme for efficient chemotherapeutic drug targeting .

about 28 million people live in areas at risk of chagas disease , 1114.5 million of whom are affected worldwide . trypanosoma cruzi , the pathogen that causes chagas disease , is found in most south american countries , representing an important cause of heart damage among the economically active population . after a successful chemical control of triatoma infestans ( klugi , 1834 ) , the other main vectors of chagas causing agent , panstrongylus megistus burmeister , 1835 , rhodnius prolixus stal , 1859 , and triatoma brasiliensis sensu lato neiva 1911 . triatoma brasiliensis sensu lato ( s.l . ) , found in anthropogenic habitats and considered the main vector in northeast brazil [ 2 , 3 ] , was recently found to be a species complex that includes t. b. brasiliensis , t. b. macromelasoma galvo , 1956 , t. juazeirensis costa & felix , 2007 , t. sherlocki papa , jurberg , carcavallo , cerqueira & barata , 2002 , and t. melanica costa et al . these taxa exhibit wide phenotypic and morphological variability , displaying specific ecological requirements and chromatic patterns . in this respect , the systematic of triatominae species is based on morphological characters of the adult exoskeleton and male phallic structures . however , insects captured during vector monitoring and control or received for identification and notification are often immature . although their characteristics are similar to those of adult individuals , they are difficult to distinguish . the morphology of triatominae species is not well described ; with studies on the immature forms performed for only 40 species , eggs and nymphs described for only 20 species , a key to identify nymphs to the species level has yet to be developed . available keys are useful and partially applicable to other stages , but specific identification of all live forms remains unresolved . members of the t. brasiliensis complex have been distinguished by analyzing isoenzymes , mitochondrial dna sequences , and random amplification of polymorphic dna - rapd . in the present study we analyzed the barcoding co1 sequences of nine t. brasiliensis s.l . populations from northeastern brazil ( states of pernambuco , paraba , and rio grande do norte ) in order to identify their genetic relationships . we also conducted a pcr - rflp assay to distinguish between t. b. macromelasoma and t. b. brasiliensis subspecies . triatominae were collected by surveillance technicians during active inspections in anthropogenic environments ( domestic and peridomestic habitats ) . live specimens were collected in nine localities in northeast brazil ( table 1 , figure 1 ) using tweezers , flashlights , and pirisa . housed in plastic boxes ( 7 cm diameter 8 cm high ) lined with folded filter paper , the bugs were transported in coolers to the culicid and triatomine laboratory of the department of epidemiology , faculty of public health / usp . adults were identified as t. b. brasiliensis according to the key by lent and wygodzinsky . nymphs were assumed to be t. brasiliensis s.l . because , in addition to the difficulty in distinguishing immature triatominae based on chromatic and morphological characters , comparative material or a key for nymph identification to species level are not available . after morphological identification , dna was extracted from individuals from the nine localities and sequenced the 520 bp barcode portion of the co1 gene ( table 1 ) . specimens collected in pernambuco state ( even nymphs ) were assumed to be t. b. macromelasoma , according to costa et al . and costa et al . . genomic dna was extracted from the legs of 10 individual samples of each population using the qiagen dneasy blood and tissue kit ( qiagen , crawley , united kingdom ) following the manufacturer 's protocol . the co1 barcode region was amplified from whole genomic dna using primers lcoi 1490 ( 5-ggt caa caa atc ata aag ata ttg g-3 ) and hcoi 2198 ( 5-taa act tca ggg tga cca aaa aat-3 ) . pcr amplification was carried out in a final volume of 50 l containing pcr buffer , 0.2 mm of each dntp , 2.5 mm mgcl2 , and 1.25 units of taq polymerase . initial pcr denaturation was at 94c for 5 min , followed by 40 cycles of denaturation ( 1 min ) at 94c , annealing ( 2 min ) at 50c , and extension ( 2 min ) at 72c . amplicons were sequenced in both forward and reverse directions using abi prism bigdye terminator cycle sequencing ready reaction kits ( perkin elmer , foster city , ca ) on an abi prism 3100 genetic analyzer / hitachi . one or two representative haplotypes for each population was chosen because of the low variation within populations . phylogenetic reconstructions were performed by neighbor joining and maximum likelihood methods ( both using the kimura-3-parameter distance model k81 ) in mega 5.0 , and a divergence matrix was constructed under kimura two parameters ( k2p ) ( table 2 ) . phylogenetic analyses included 1000 bootstrap replicates and a triatoma sordida co1 sequence ( genbank acc . co1 sequences were also analyzed using nebcutter version 2.0 to select appropriate endonuclease enzymes . individual co1 sequences were pcr - amplified using the above parameters and digested in a 10 l reaction with styi ( promega ) and hincii ( new england biolabs , ipswich , ma ) enzymes . the reaction contained 1 l of 10x buffer , 4 l deionized water , 4 l of amplification product , and 1 unit of restriction enzyme . the digestion mixture was incubated at 37c for 2 h and then resolved on 2.0% agarose gel . phylogenetic trees derived from the neighbor joining , maximum likelihood , and parsimony methods showed similar topologies ( data shown for ml tree in figure 2 ) . since most of co1 sequences were identical into and among the populations , the ml tree was constructed using only two samples of each one . this ml tree and the nucleotide distance matrix ( table 2 ) indicated sequence divergence of up to 4% between the two main clades . the basal clade , with about 4% divergence from the other populations , consisted of the salgueiro population ( 15a / b ) . this population was considered to be t. b. macromelasoma due to its high sequence divergence compared to the other populations and because it was collected in its type locality . the second clade , with pairwise distances up to 1% , showed that pernambuco populations are more basal , although those from serra talhada clustered with paraba populations likely because of the city 's proximity to the pernambuco - paraba border . paraba populations formed a large cluster that also included the rio grande do norte population . based on sequence analysis , a pcr - rflp assay was performed to differentiate between the subspecies t. b. macromelasoma and t. b. brasiliensis . pcr fragment digestion using the styi enzyme produced two restriction fragments ( 342 bp and 192 bp ) in co1 sequences from salgueiro samples and only one fragment in samples of the other eight populations . conversely , the hincii enzyme yielded two fragments ( 297 bp and 240 bp ) in all population samples ( n = 10 samples of each population ) except that from salgueiro ( figure 3 ) . styi and hincii enzymes therefore produced distinct fingerprints for t. b. macromelasoma and t. b. brasiliensis , suggesting that they are different subspecies . the molecular protocols described above are a useful tool in the study of populations and cryptic species , contributing to the identification of insect vectors . the identification of adult triatominae based on morphological and chromatic pattern is considered relatively easy for most species ; however , this is commonly misguided owing to the wide phenotypic variability within this subfamily . for instance , triatoma maculata erichson , 1848 and triatoma pseudomaculata corra & espnola , 1964 which were first treated as members of a same species complex due to morphological similarities thereafter proved to be genetically distant [ 1719 ] . panstrongylus herreri wygodzinsky , 1948 and panstrongylus lignarius walker , 1873 in turn , were considered to be separate species until marcilla et al . and crossa et al . demonstrated that they are the same species , cytogenetically identical with regard to the second internal transcribed spacer . ( light brown tones ) and rhodnius nasutus stl , 1859 ( pinkish tones ) seems to be associated with the color of the palm trees they colonize , but others such as triatoma rubrovaria blanchard , 1843 , exhibit well - known 4 chromatic morphotypes . other studies report the occurrence of natural homoploid hybrids between t. infestans and triatoma platensis neiva , 1913 , t. infestans and triatoma rubrovaria , and sympatric species of phyllosoma complex and species of the t. brasiliensis complex . this interspecific crossing can be decisive in originating and diversifying wild species , resulting in important epidemiological consequences due to differential competence and the capacity of hybrid vectors [ 3 , 10 , 11 ] . therefore , the characterization ( or identification ) of triatominae specimens based only on morphological and chromatic patterns , the most common identification method , is more complex than previously believed . studies on immature stages are crucial for group systematics . however , literature reports on immature forms of certain groups are scarce , difficult to use , or nonexistent . many species undergo changes in color , structure , and morphology during their development , hindering their identification [ 25 , 26 ] . , molecular analyses are successfully used to characterize morphotypes of species complexes such as t. brasiliensis sp . , which exhibits wide chromatic and morphological variation [ 7 , 8 ] . marked differences in color pattern and ecological features among species from the triatoma brasiliensis complex were detected by microsatellites , mitochondrial 12s , and cytochrome b genes , reinforcing species diagnosis [ 7 , 16 ] . however , individuals from subspecies t. b. brasiliensis and t. b. macromelasoma might be clustered within the same co1 clade , since earlier studies have shown that some cytochrome b haplotypes of t. b. macromelasoma are similar to those of t. b. brasiliensis . moreover , the basal clade of the maximum likelihood tree ( figure 1 ) was identified as subspecies t. b. macromelasoma because it is highly divergent ( 6% , as shown in the nucleotide distance matrix , table 2 ) from the other populations and was collected in its type locality . on the other hand , however , interpopulation divergence values ( < 1% ) suggest that t. b. brasiliensis is still diversifying and/or exhibiting ongoing gene flow , probably due to human - assisted dispersal . based on wing morphometry , costa et al . recently formulated a hypothesis that t. b. macromelasoma is the result of homoploidal hybridization between t. b. brasiliensis and t. juazeirensis in the state of pernambuco , and that this is a form of speciation in sympatric populations . in northeastern brazil , the epidemiological importance of triatominae bugs is mainly defined by their high rate of natural t. cruzi infection and ability to adapt to multiple ecotopes . control measures therefore require a precise identification of which species of the t. brasiliensis complex is being targeted . moreover , it is important to understand the ecoepidemiology of triatominae since these vectors are found in large numbers in their natural habitat . in this respect , the pcr - rflp protocol described here is suggested as rapid , relatively simple , and economical assay to distinguish triatoma b. macromelasoma from triatoma b. brasiliensis subspecies . even at small geographic scales , domestic populations are genetically structured by ecological parameters , thereby exhibiting small differences from the wild counterparts from which they are derived . the present study highlights the effectiveness of the co1 gene in identifying subspecies of the t. brasiliensis complex and its contribution to classic taxonomy . ablation of the cavotricuspid isthmus ( cti ) for the treatment of atrial flutter ( afl ) has become standard practice . most of the procedures are performed using radiofrequency energy ( rf ) [ 15 ] . high chronic success rates are described but the majority of the data comes from a relatively short term follow - up [ 113 ] . a recent study by chinitz et al . reported some interesting data regarding the long term follow - up of 80 patients with common type afl who underwent cti ablation using rf . they found a 12.5% ( ten patients ) recurrence rate at an average of 21 months after the procedure with most patients having a recurrence after the first year post ablation . our prior experience using cryothermy in a limited number of patients also showed that afl may recur 1 year after cti cryoablation . the purpose of this study was to evaluate the long term outcome of cti cryoablation in a large patient population with common type afl in a single center . one hundred and eighty patients with sustained symptomatic common type afl referred for cti ablation were enrolled prospectively from july 2001 to july 2006 in our institution . signed written consent , approved by the local ethics committee , was obtained from all participants . before cti cryoablation , anticoagulation with warfarin aiming a therapeutic international normalized ratio of 2 to 3 was kept for at least 3 weeks . we focused our study on the clinical aspects and long term follow - up of patients with afl who were submitted to cti cryoablation . briefly , our methodology was as follows : under local anesthesia and via the femoral route , a decapolar catheter is positioned in the distal coronary sinus ( for evaluation of left atrial activation ) , a duodecapolar catheter ( 2-mm interelectrode spacing , halo catheter , biosense webster , baldwin park , ca ) for mapping the right atrial lateral wall and a quadripolar catheter in the his bundle area . a deflectable , 10.5 f , 6.5 mm tip cryoablation catheter ( cryocor inc . , san diego , ca ) is inserted into the right atrium through a 12f , 65-cm - long sheath ( daig , st . entrainment to confirm the isthmus dependence of the afl circuit was performed in every patient in whom afl was present or could be induced at the start of the procedure , according to previously published techniques [ 2 , 10 , 15 , 16 , 20 , 21 ] . if af , requiring cardioversion , was present or developed during stimulation or if we were unable , even under isoproterenol infusion , to induce afl , cti ablation was performed during sinus rhythm . linear lesions were created by use of a point - by - point technique with gradual pullback of the cryocatheter in a ventricular atrial fashion . the first application was delivered at the ventricular insertion of the isthmus and applications were continued for an average of 3 min . in patients in whom ablation of the posterior isthmus proved insufficient , an attempt was made to ablate the septal isthmus ( four patients ) . after documentation of bidirectional isthmus conduction block , the atrial pacing ( from the proximal coronary sinus ) protocol ( up to three atrial extrastimuli at three pacing cycle lengths and incremental atrial pacing ) was performed without and with isoproterenol infusion ( 1 to 3 g / min ) . acute success was defined as bidirectional isthmus conduction block , 30 min after the last application without and with isoproterenol infusion . those presenting in af while on the catheterization table were converted to sinus rhythm by internal or external cardioversion . during the procedure intravenous heparin was given as a 100-iu / kg bolus dose after the venous sheaths were inserted . the 12-lead ecg and intracardiac electrograms were recorded and stored by the bard labsystem pro . all patients were monitored in hospital for 24 h and oral anticoagulation was started the day of ablation . antiarrhythmic drugs ( aad ) were stopped after the procedure in patients without a history of af ; in those with af / afl the same aad were continued . subsequently , the need for chronic anticoagulation was assessed by the amount of recurrences of afl / af and the presence of risk factors for thromboembolic events . all patients had a 12-lead ecg and a 24 h holter recording at discharge and during each clinic visit ( 1 month , 3 , 6 , 9 , 12 months and yearly thereafter ) or earlier if they had symptoms . due to the logistics of the maastricht area and also the presence of a dedicated research nurse ( s. p. ) who was available to address patients concerns and questions at any time we were able to follow every patient on an individual basis . continuous variables are presented as mean sd , where appropriate . in cases of a non - gaussian distribution , categorical variables are expressed as numbers and percentages of patients . statistical analysis was performed using the student t test for unpaired data . we focused our study on the clinical aspects and long term follow - up of patients with afl who were submitted to cti cryoablation . briefly , our methodology was as follows : under local anesthesia and via the femoral route , a decapolar catheter is positioned in the distal coronary sinus ( for evaluation of left atrial activation ) , a duodecapolar catheter ( 2-mm interelectrode spacing , halo catheter , biosense webster , baldwin park , ca ) for mapping the right atrial lateral wall and a quadripolar catheter in the his bundle area . a deflectable , 10.5 f , 6.5 mm tip cryoablation catheter ( cryocor inc . , san diego , ca ) is inserted into the right atrium through a 12f , 65-cm - long sheath ( daig , st . entrainment to confirm the isthmus dependence of the afl circuit was performed in every patient in whom afl was present or could be induced at the start of the procedure , according to previously published techniques [ 2 , 10 , 15 , 16 , 20 , 21 ] . if af , requiring cardioversion , was present or developed during stimulation or if we were unable , even under isoproterenol infusion , to induce afl , cti ablation was performed during sinus rhythm . linear lesions were created by use of a point - by - point technique with gradual pullback of the cryocatheter in a ventricular atrial fashion . the first application was delivered at the ventricular insertion of the isthmus and applications were continued for an average of 3 min . in patients in whom ablation of the posterior isthmus proved insufficient , an attempt was made to ablate the septal isthmus ( four patients ) . after documentation of bidirectional isthmus conduction block , the atrial pacing ( from the proximal coronary sinus ) protocol ( up to three atrial extrastimuli at three pacing cycle lengths and incremental atrial pacing ) was performed without and with isoproterenol infusion ( 1 to 3 g / min ) . acute success was defined as bidirectional isthmus conduction block , 30 min after the last application without and with isoproterenol infusion . those presenting in af while on the catheterization table were converted to sinus rhythm by internal or external cardioversion . during the procedure intravenous heparin was given as a 100-iu / kg bolus dose after the venous sheaths were inserted . the 12-lead ecg and intracardiac electrograms were recorded and stored by the bard labsystem pro . all patients were monitored in hospital for 24 h and oral anticoagulation was started the day of ablation . antiarrhythmic drugs ( aad ) were stopped after the procedure in patients without a history of af ; in those with af / afl the same aad were continued . subsequently , the need for chronic anticoagulation was assessed by the amount of recurrences of afl / af and the presence of risk factors for thromboembolic events . all patients had a 12-lead ecg and a 24 h holter recording at discharge and during each clinic visit ( 1 month , 3 , 6 , 9 , 12 months and yearly thereafter ) or earlier if they had symptoms . due to the logistics of the maastricht area and also the presence of a dedicated research nurse ( s. p. ) who was available to address patients concerns and questions at any time we were able to follow every patient on an individual basis . continuous variables are presented as mean sd , where appropriate . in cases of a non - gaussian distribution , of the 180 enrolled patients , 39 patients ( 22% ) were women with a mean age of 58 ( from 18 to 80 ) years . more than half of the patients ( 92 patients , 52% ) had structural heart disease : arterial hypertension : 57 patients , coronary artery disease : 22 patients , valvular heart disease : 13 patients , congenital heart disease : 11 patients , idiopathic cardiomyopathy : 18 patients . counterclockwise afl was documented in 91% ( 164 ) of the patients and clockwise afl in 9% ( 16 patients ) . the mean left atrial diameter and the mean left ventricular ejection fraction were 44 7 mm and 57 7% , respectively . a prior history of af was present in 123 ( 69% ) of the patients . the clinical characteristics of the patients , related to the presence or absence of af before ablation , are described in table 1 . note that af during follow - up is significantly higher in the group with a prior history of this arrhythmia . table 1characteristics of the 180 patients with atrial flutter referred for cti cryoablation related to the presence or absence of atrial fibrillation af before ablation af / afl patients ( 123 patients ) , 69%afl only ( 57 patients ) , 31%p valueage ( year)57 1358 13nswomen19% ( 23 patients)28% ( 16 patients)nsno shd55% ( 68 patients)32% ( 18 patients ) < 0.05lad ( cm)4.44.5nslvef ( % ) 5855nsacute failures5% ( 6 patients)5% ( 3 patients)nsaf in long term follow up69% ( 85 patients)35% ( 20 patients ) < 0.05patients in whom cti cryoablation did not result in bidirectional block ( failed procedure).af atrial fibrillation , afl atrial flutter , cti cavotricuspid isthmus , lad left atrium diameter , lvef left ventricular ejection fraction , shd structural heart disease characteristics of the 180 patients with atrial flutter referred for cti cryoablation related to the presence or absence of atrial fibrillation af before ablation patients in whom cti cryoablation did not result in bidirectional block ( failed procedure ) . af atrial fibrillation , afl atrial flutter , cti cavotricuspid isthmus , lad left atrium diameter , lvef left ventricular ejection fraction , shd structural heart disease total fluoroscopic mean of 30 18 min ( range , 12 to 152 min)and procedure times mean of 2.6 1.1 h ( range , 1 to 6.5 h)decreased throughout our study with a long duration of a procedure and fluoroscopy being attributed mainly to the learning curve of a new technology . an average of 7 ( 3 to 20 ) applications per patient were delivered with a mean temperature and duration of 88c and 3 min , respectively . the acute success rate for cryoablation of the cti was 95% ( 171 patients ) . of the nine patients in whom bidirectional cti block was not achieved , three underwent a successful reablation . the other six patients had much improvement of their symptoms ( despite an incomplete line ) and preferred not to have another procedure . after a mean follow - up of 27 17 ( range from 12 to 60 ) months , recurrences of symptomatic afl occurred in 15 patients ( 9% ) resulting in a 91% chronic success rate . those recurrences occurred in six patients within the first 3 months post ablation , in four patients from 3 to 6 months post ablation , in four patients from 6 to 9 months post ablation and in one patient at 14 months post ablation ( fig . 1 ) . the other five had not only reablation of the isthmus but also pulmonary vein cryoisolation ( pvi ) for af during the second procedure . 1percentage of patients ( 171 successfully ablated ) free of common type atrial flutter over time percentage of patients ( 171 successfully ablated ) free of common type atrial flutter over time despite the success as far as afl was concerned , af was still present in 85 patients ( 69% ) with a prior history of this arrhythmia . those patients were treated by aad ( 69 patients , 81% ) , pvi ( 14 patients , 16% ) or av nodal ablation with pacemaker implantation ( two patients , 3% ) . new episodes of af developed in 20 ( 35% ) of those 57 patients without documented af prior to cti cryoablation and were all controlled by aad . our current study showed a 91% chronic success rate of cti cryoablation in a large population ( 180 patients ) with afl followed for a long period of time ( 1 to 5 years , mean of 27 months ) . according to the most recent acc / aha / esc guidelines for the management of supraventricular arrhythmias cti ablation is the only therapy with a class i indication for the long term management of afl . the majority of those ablations are done using rf energy . concerning the follow - up length of those procedures , most of the literature available reports on a relatively short period , the majority of them being less than 2 years . however chinitz et al . published a study of 80 patients with afl submitted to cti ablation using rf that had up to 6 years follow - up . interestingly , they found a 12.5% recurrence of afl that occurred on an average of 21 months post ablation , ratifying the need of data with a longer follow - up . it is important to keep in mind though , that arrhythmias after ablation do not always correlate with symptoms [ 23 , 24 ] and one could question if those patients with such a late recurrence had those episodes much earlier than what is reported . that might be one reason why our results , where most of our recurrences happened within 1 year , are discrepant with those from their study . the intrinsic characteristics of our hospital , our clinical follow - up and the population of maastricht could be responsible for our ability to find those recurrences earlier . regarding cryothermy as energy source , a 9 months follow - up study in 39 patients undergoing cti cryoablation with a different system was reported . they achieved a chronic success rate of 100% in the cryoablated group , despite documenting reconduction through the isthmus in 31% of patients during a 3 month follow - up electrophysiologic study . the craaft trial presented the results in 32 patients with afl submitted to either cryo- or rf ablation of the cti . they report an 84% success rate after a follow - up of 14 months . those two small studies , with a relatively short follow - up , reported similar success rates with rf and cryothermy for afl ablation . the results from our study which included a significant larger patient population ( 180 ) with a longer follow - up ( 1 to 5 years , mean of 27 months)reinforces the effectiveness of cryothermy for the treatment of afl , being the outcomes comparable to those reported in the literature using rf ( where most outcome data also comes from non invasive follow - up ) [ 1 , 47 , 11 , 12 , 27 , 28 ] . therefore , cryothermy can be considered as an efficient energy source for the treatment of common type afl . the close relation between af and afl our data showed a high prevalence of af ( 123 out of 180 patients , 69% ) in patients with predominant afl referred for cti ablation . a recent study by ellis and colleagues strengthened even more this association . they followed 363 patients with lone afl who underwent cti rf ablation during a mean follow - up of 39 months and reported an 82% incidence of drug refractory af in their patient population . the new development of af in our patient population without a prior history of af preceding afl ablation may be a sign of an already present electrical and morphological change in the right and left atria . if in addition , a functional or anatomical line of block between the two venae cavae ( or elsewhere ) occurs , atypical afl(s ) may develop either in the right ( because the cti is already ablated ) or in the left atrium [ 11 , 14 , 21 , 31 , 32 , 3739 ] . our recurrence data rely mostly on the subjective assessment by the patient , like the great majority of rf data [ 1 , 4 , 68 , 12 ] . only an objective measurement ( such as a repeat electrophysiological study with documented bidirectional block ) will determine the long lasting effect of cti ablation for the treatment of common type afl . our current study showed a 91% chronic success rate of cti cryoablation in a large population ( 180 patients ) with afl followed for a long period of time ( 1 to 5 years , mean of 27 months ) . according to the most recent acc / aha / esc guidelines for the management of supraventricular arrhythmias cti ablation is the only therapy with a class i indication for the long term management of afl . the majority of those ablations are done using rf energy . concerning the follow - up length of those procedures , most of the literature available reports on a relatively short period , the majority of them being less than 2 years . however chinitz et al . published a study of 80 patients with afl submitted to cti ablation using rf that had up to 6 years follow - up . interestingly , they found a 12.5% recurrence of afl that occurred on an average of 21 months post ablation , ratifying the need of data with a longer follow - up . it is important to keep in mind though , that arrhythmias after ablation do not always correlate with symptoms [ 23 , 24 ] and one could question if those patients with such a late recurrence had those episodes much earlier than what is reported . that might be one reason why our results , where most of our recurrences happened within 1 year , are discrepant with those from their study . the intrinsic characteristics of our hospital , our clinical follow - up and the population of maastricht could be responsible for our ability to find those recurrences earlier . regarding cryothermy as energy source , a 9 months follow - up study in 39 patients undergoing cti cryoablation with a different system was reported . they achieved a chronic success rate of 100% in the cryoablated group , despite documenting reconduction through the isthmus in 31% of patients during a 3 month follow - up electrophysiologic study . the craaft trial presented the results in 32 patients with afl submitted to either cryo- or rf ablation of the cti . they report an 84% success rate after a follow - up of 14 months . those two small studies , with a relatively short follow - up , reported similar success rates with rf and cryothermy for afl ablation . the results from our study which included a significant larger patient population ( 180 ) with a longer follow - up ( 1 to 5 years , mean of 27 months)reinforces the effectiveness of cryothermy for the treatment of afl , being the outcomes comparable to those reported in the literature using rf ( where most outcome data also comes from non invasive follow - up ) [ 1 , 47 , 11 , 12 , 27 , 28 ] . therefore , cryothermy can be considered as an efficient energy source for the treatment of common type afl . the close relation between af and afl is well described [ 1 , 514 , 21 , 2939 ] . our data showed a high prevalence of af ( 123 out of 180 patients , 69% ) in patients with predominant afl referred for cti ablation . a recent study by ellis and colleagues strengthened even more this association . they followed 363 patients with lone afl who underwent cti rf ablation during a mean follow - up of 39 months and reported an 82% incidence of drug refractory af in their patient population . the new development of af in our patient population without a prior history of af preceding afl ablation may be a sign of an already present electrical and morphological change in the right and left atria . if in addition , a functional or anatomical line of block between the two venae cavae ( or elsewhere ) occurs , atypical afl(s ) may develop either in the right ( because the cti is already ablated ) or in the left atrium [ 11 , 14 , 21 , 31 , 32 , 3739 ] . our recurrence data rely mostly on the subjective assessment by the patient , like the great majority of rf data [ 1 , 4 , 68 , 12 ] . only an objective measurement ( such as a repeat electrophysiological study with documented bidirectional block ) will determine the long lasting effect of cti ablation for the treatment of common type afl . in this prospectively studied large population of patients with common type afl , cryoablation of the cti has a 91% chronic success rate during long term follow - up ( range 1 to 5 years , mean of 27 months ) . these results are similar to those obtained with rf , validating cryothermy as an efficient alternative energy source .

triatoma brasiliensis sensu lato ( s.l . ) , the main vector of chagas disease in northeastern brazil , is a species complex comprising four species , one with two subspecies ( t. brasiliensis brasiliensis , t. brasiliensis macromelasoma , t. juazeirensis , t. sherlocki , and t. melanica ) , and each taxon displaying distinct ecological requirements . in order to evaluate the genetic relationships among nine t. brasiliensis s.l . populations from northeastern brazil , we analyzed their mitochondrial cytochrome c oxidase subunit 1 sequences and suggested a pcr - rflp assay to distinguish between t. b. macromelasoma and t. b. brasiliensis subspecies . all the specimens were morphologically identified as t. b. brasiliensis . the resulting phylogenies identified two major clades that are congruent with the geographical populations studied . based on collection sites and in accordance with type - location , one clade was identified as the subspecies t. b. macromelasoma . the second clade grouped t. b. brasiliensis populations . restriction endonuclease sites were observed in the sequences and used in pcr - rflp assays , producing distinct fingerprints for t. b. macromelasoma and t. b. brasiliensis populations . the results suggest that these are different species and that gene flow occurs only among t. b. brasiliensis populations , possibly associated with human activity in the area . objectiverecent literature has shown that common type atrial flutter ( afl ) can recur late after cavotricuspid isthmus ( cti ) catheter ablation using radiofrequency energy ( rf ) . we report the long term outcome of a large group of patients undergoing cti ablation using cryothermy for afl in a single center.methodspatients with afl referred for cti ablation were recruited prospectively from july 2001 to july 2006 . cryoablation was performed using a deflectable , 10.5 f , 6.5 mm tip catheter . cti block was reassessed 30 min after the last application during isoproterenol infusion . recurrences were evaluated by 12-lead ecg and 24 h holter recording every clinic visit ( 1/3/6/9 and 12 months after the procedure and yearly thereafter ) or if symptoms developed.resultsthe 180 enrolled patients had the following characteristics : 39 women ( 22% ) , mean age 58 years , no structural heart disease in 86 patients ( 48% ) , mean left atrium diameter 44 7 mm and mean left ventricular ejection fraction 57 7% . the average number of applications per patient was 7 ( 3 to 20 ) with a mean temperature and duration of 88c and 3 min , respectively . acute success was achieved in 95% ( 171 ) of the patients . there were no complications . after a mean follow - up of 27 17 ( from 12 to 60 ) months , the chronic success rate was 91% . the majority of the recurrences occurred within the first year post ablation . one hundred and twenty three patients had a history of atrial fibrillation ( af ) prior to cti ablation and 85 ( 69% ) of those remained having af after cryoablation . in 20 of 57 ( 35% ) patients without a history of af prior to cti ablation , af occurred during follow-up.conclusionsthis prospective study showed a 91% chronic success rate ( range 12 to 60 months ) for cryoablation of the cti in patients with common type afl and ratified the frequent association of af with afl .

an anal fistula is a chronic phase of anorectal sepsis and is characterized by chronic purulent drainage or cyclical pain associated with abscess formation , followed by intermittent spontaneous decompression [ 1 , 2 ] . the goals in the treatment of an anal fistula are to eliminate the primary fistula opening , any associated tracts , and any secondary openings without a change in continence . most anal fistulae are simple and can be treated using a fistulotomy , which has a low recurrence rate and an acceptable rate of morbidity [ 3 - 6 ] . however , the treatment of a complex anal fistula , which is defined as a fistula whose treatment poses an increased risk for a change in continence , still represent a challenge [ 7 - 9 ] . the recurrence rate for a complex anal fistula managed with a cutting seton is reported to be 0 to 8% , with minor and major incontinence being reported in 34 to 63% and 2 to 26% of patients , respectively [ 10 - 14 ] . advancement flap is still considered to be the gold standard of treatment for a complex anal fistula . successful healing of the fistula has been demonstrated in 55 to 98% of patients [ 8 , 10 , 15 - 17 ] . however , this procedure is technically demanding , and although the sphincter mechanism is not divided during advancement flap repair of the fistula , minor incontinence has been found in up to 31% patients and major incontinence in up to 12% of patients [ 10 , 18 , 19 ] . because of the risk of a change in continence with these conventional techniques , sphincter - preserving techniques for the management of complex anal fistulae have been evaluated . glue was easy to apply , but probably not ideal for fistula treatment because of its liquid consistency . a failure of the formed glue clot in a properly sealed tract , the inability to securely fix the material within the tract and the uncertainty of tissue in growth into the glue may all explain the possible causes of the poor outcomes . this article aims to review the literature and to identify the new sphincter - preserving techniques , such as the anal fistula plug , the ligation of intersphincteric fistula tract ( lift ) procedure and the cell therapy , used in the management of anal fistulae . the small intestinal submucosa is a natural biomaterial harvested from porcine small intestine and fabricated into a biomedical product of various shapes and thickness . the fact that it has been demonstrably useful as a bioprosthetic material in infected fields makes its application in fistula surgery quite reasonable . 1 ) . the idea is to bridge the defect of the fistula with a biocompatible material that would act as a scaffold for the patient 's own fibroblasts to come in and promote wound healing in the fistula tract [ 23 , 24 ] . the technique of plug deployment is as follows : the tract is explored , probed , and irrigated gently with hydrogen peroxide . then , the apex of the plug is tied to the probe from the internal opening , and the plug is dragged through to the external opening . it is cut to fit and is secured in the internal opening by using a figure - of - eight suture , incorporating it with the mucosa of the anorectum to close the internal opening ( fig . the anal fistula plug has been used in a number of cases with widely varying results ( table 1 ) [ 21 , 24 , 26 - 33 ] . in an early prospective series of 46 patients reported by champagne et al . , after a median follow - up of 12 months ( range , 6 to 24 months ) , 17% of fistulae recurred . johnson et al . published a series comparing two prospective cohort groups of patients undergoing plug closure versus patients undergoing fibrin glue closure . they reported an 87% closure rate for the plug group versus a 40% closure rate for the glue group . others have had less favorable results with fistula recurrence rates as high as 80% [ 21 , 28 - 31 ] . on review of these publications , possible explanations for this discrepancy include differences in patient selection and technical problems . in an attempt to standardize the indications for use of the bioprosthetic anal fistula plug and techniques for its placement , first , the group stressed that the use of the fistula plug could only be recommended for use in transsphincteric anal fistulae without acute inflammation or infection . the group felt that dislodgement of the anal fistula plug was a problem related to issues in the technique of plug placement . the group also stressed the importance of the external fistula opening being patent to allow for drainage . although most fistula recurrences occur within the first 3 months after treatment , a significant number of recurrences are known to occur after this time . a minimum one - year follow - up is generally accepted as being needed to justify the results of sphincter - preserving surgery . reported that posterior fistula location , tobacco smoking , and a history of previous failure of the plug were independent predictors of plug failure . the main limitation of application of the anal fistula plug in the treatment of anal fistulae in korea is the high cost . in korea , the cost of an anal fistula plug is not reimbursed by the national insurance system . therefore , the cost of using a plug must be paid by the patients themselves . it is interesting to note that the anal fistula plug procedure would probably be suitable for a low transsphincteric fistula . in a study from the university of minnesota , however , it was 65% for fistulae that involved less than one - third of the sphincter and 22% for those involving two - thirds or more . recent reports from chulalongkorn university , bangkok , thailand , have described a novel technique called lift for the treatment of anal fistulae . a success rate of 94.4% was reported in the treatment of 18 patients [ 34 , 35 ] . this technique prevents the entry of fecal material into the fistula tract and eliminates the formation of a septic nidus in the intersphincteric space to allow healing of the anal fistula . the location of internal opening is identified by injection of hydrogen peroxide or water through the external opening or by gently probing the fistula tract . a 1.5 to 2.0 cm curvilinear incision is made at the intersphincteric groove overlying the fistula tract . the dissection is kept close to the external sphincter to avoid cutting through the internal sphincter and breaching the anal mucosa . after the intersphincteric tract has been identified and dissected out , the tract is ligated close to the internal sphincter . . the tract next to the suture site is divided , and the rest of intersphincteric tract is excised . after removal of the correct fistulous tract has been confirmed , infected granulation tissues in the rest of the tract and cavity are thoroughly removed with curettage . the lift procedure has been used in five case series with promising early results ( table 2 ) [ 34 , 36 - 39 ] . bleier et al . reported that successful fistula closure was achieved in 57% of the 35 patients . the advantages of the lift procedure may include preservation of the anal sphincter , minimal tissue injury , shorter healing time , and its being a procedure that is relatively easy to perform . additionally , even if the fistula is not healed successfully , the lift procedure may convert a difficult - to - treat transsphincteric fistula into an easier - to - manage intersphincteric fistula . first , the published results are the only case series of a heterogeneous population , and the good results may come from selection bias . second , there were some variations in the manner in which the fistula was ligated and in the coring out or curettage of the external tract . third , a longer and objective follow - up would be a better indicator of the durability , and no standardized questionnaires were used pre- or postoperatively with respect to fecal incontinence and quality of life . additionally , as is the anal fistula plug , the indication for the lift procedure seems to be limited to the transsphincteric fistula . interestingly , another intersphincteric approach for the treatment of a complex anal fistula has been described by matos et al . . first , the ligation of the fistula tract is more secure than over sewing , and second , removal of infected granulation tissue by curettage is more practical and less time - consuming than total excision of the tract and primary repair . apart from the difficulty in the lift procedure for a high transsphincteric or a suprasphincteric fistula , the identification of the intersphincteric tract through the intersphincteric approach seems to require more advanced technical skills than the removal of the intersphincteric tract and infected granulation tissue in a conventional technique does . neal ellis published outcomes with the biolift technique for the management of transsphincteric fistulae . the biolift technique is a variation of the lift technique in which a bioprosthetic is placed in the intersphincteric plane to reinforce the closure of the fistula tract . the bioprosthetic graft acts as a physical barrier in the intersphincteric space . actually , the biolift technique utilized a transection of the intersphincteric tract and closure of the fistula opening in the internal sphincter , which is similar to the procedure described by matos et al . , instead of ligating the intersphincteric tract . when compared to the lift , the biolift technique has two potential disadvantages . first , it requires a more extensive dissection in the intersphincteric space because the bioprosthetic must overlap the closure of the fistula tract by at least 1 to 2 cm in all directions . mesenchymal adult stem cells extracted from certain tissues , such as adipose tissue , can differentiate into various types of cell . [ 43 , 44 ] chose adipose tissue as the source of stem cells because of their two biologic properties : their ability to suppress inflammation and their differentiation potential . moreover , enough fat can be obtained with liposuction , and the fat cells can be harvested with minimal adverse effects on the patient . their phase i clinical trials show that adipose - derived stem cells are safe for the treatment of a fistula in crohn 's disease , and they published a phase ii multicenter , randomized controlled trial to further investigate the effectiveness and safety of ascs in the treatment of complex anal fistulae compared with the fibrin glue group . the treatment procedures are as follows : 1 ) tract identification , with special emphasis on the identification of the internal opening ; 2 ) tract curettage , with special emphasis on the intersphincteric tract ; 3 ) closure of the internal opening ; 4 ) injection of a cell suspension through a long fine needle into the tract walls ; 5 ) sealing of the tract with fibrin glue . they reported that fistula healing was observed in 71% of 24 patients who received ascs in addition of fibrin glue compared with 16% of 25 patients who received fibrin glue alone ( relative risk for healing , 4.43 ; confidence interval , 1.74 to 11.27 ; p < 0.001 ) . the proportion of patients with healing was similar between the crohn 's and the non - crohn 's subgroups . an advantage of using stem cells to treat an anal fistula is that , because tract resection is not required , the treatment does not injure the anal sphincter ( fig . 4 ) . there are , nonetheless , some limitations of the ascs - based therapy . first , the cost of treatment with ascs is difficult to evaluate in detail at present . second , technically , secure closure of the internal opening and complete injection of the cell suspension over the tract may be complicated procedures . finally , as yet , there are no reports on the long - term effects of using ascs . in the meantime , a pilot study on autologous fibroblast - based therapy for the treatment of anal fistulae is in progress in the united kingdom . in brief , the authors suggest that the addition of autologous fibroblasts to the filling material ( collage paste ) provides the best histologic healing . to the colorectal surgeon , the complex anal fistula remains a challenging condition to manage despite the best of technologic advances . therefore , the colorectal surgeon should become familiar with various new techniques for treating an anal fistula and remember that conventional fistula surgery , such as cutting seton and advancement flap , has an important role . especially , the advancement flap is still the gold standard of treatment for complex anal fistulae . definitive evidence of the advantage of the new sphincter - preserving techniques compared with the traditional intervention requires randomized control studies , which are now being conducted . a growing body of evidence demonstrates that susceptibility and progression of both acute and chronic central nervous system ( cns ) disease is closely associated with an innate immune response that can manifest from either direct infection and/or infection - triggered damage . a common feature of these diseases is the systemic activation of inflammatory mediators , which via the blood can disrupt the blood - brain barrier , affect the circumventricular organs in the brain ( which lack a blood - brain barrier ) , or interact with the brain endothelium , thereby eliciting brain inflammation . furthermore , the presence of activated inflammatory cells derived from systemic circulation or from dormant brain resident populations is a key feature of many cns diseases . more recently , the importance of innate immune receptors in cns injury , the so - called toll - like receptors ( tlrs ) , has also been emphasized . in this paper we will focus on how neonatal sepsis and tlr - mediated inflammation increase the vulnerability of the newborn brain . infants with sepsis have an increased incidence of cerebral palsy and white matter abnormalities [ 511 ] . in a large study of 6093 extremely low birth weight ( < 1000 g ) infants , those who were infected ( including early - onset sepsis , suspected sepsis ( culture negative ) , and had necrotizing enterocolitis ( nec ) ) were more likely to have cerebral palsy than children who did not have a neonatal infection . in another recent large sample - size study involving 1155 infants born at 23 to 27 weeks gestation , it was found that children who had both late bacteremia ( positive blood culture result after the first postnatal week ) and surgical nec were at increased risk of diparetic cerebral palsy compared with children who had neither . moreover , by comparing outcomes of 150 infants with periventricular leukomalacia ( pvl ) with controls matched for gestational age , it was found that infants with bacterial sepsis were twice as likely to develop pvl , and those with meningitis were almost four times as likely to develop white matter disease . similar findings were noted in a smaller case - control study , where associations between cerebral palsy , clinical chorioamnionitis and sepsis were demonstrated . moreover , there was an increased incidence of gram - negative bacterial and fungal infections in a very low birth weight population , and these infants were at significantly increased risk for moderate to severe cerebral palsy and neurodevelopmental impairment at 18 months of age . escherichia coli is one of the main pathogens causing early - onset infections in preterm neonates , accounting for up to 40% of the cases of bacteremia among very low birth weight preterm infants ( < 1,500 g ) . cerebral white matter injury has been found by mri following escherichia coli meningitis in human newborn infants . furthermore , escherichia coli induce brain damage in a number of antenatal rabbit and rodent models [ 1926 ] . also , in a recent study , white matter injury was demonstrated in an animal model of neonatal escherichia coli sepsis in 5-day - old rat pups . experimental studies show that early - life escherichia coli exposure can also have long - term effects , influencing the vulnerability to other factors in adulthood , for example , age - related cognitive decline as well as attenuated glial and cytokine responses to amphetamine challenge . in recent years , coagulase - negative staphylococci ( cons ) have emerged as the most prevalent and important neonatal pathogens , responsible for approximately 50% of all episodes of late - onset neonatal sepsis in neonatal intensive care units around the world [ 3033 ] . cons cause significant morbidity , mortality , and healthcare costs worldwide in preterm newborns , especially in very low birth weight infants [ 3438 ] . the vulnerability of preterm infants to cons infection has been suggested to be due to the special characteristics of the premature infant 's innate immunity . although there is no direct evidence of cons causing perinatal brain injury , the presence of cons in the chorioamnion space at delivery is associated with increased risk for the development of cerebral palsy in preterm infants [ 40 , 41 ] . further , in children with an established diagnosis of cerebral palsy , who are admitted to pediatric intensive care , there is a high rate of carriage of abnormal bacteria , including cons . in very low birth weight preterm infants with early onset neonatal sepsis , the rate of group b streptococcal ( gbs ) infections is relatively low in comparison with e. coli infections . there is no direct evidence of gbs sepsis playing a role in cerebral palsy ; however , nearly half of all infants who survive an episode of gbs meningitis suffer from long - term neurodevelopmental sequelae . further , extensive cortical neuronal injury was found in gbs - infected neonatal rats , which was mediated through reactive oxygen intermediates [ 44 , 45 ] . toll - like receptors ( tlrs ) play a central role in primary recognition of infectious and viral pathogens . the presence of all 13 known tlrs has been demonstrated in the brain [ 4648 ] . tlr4 mediates cellular activation in response to lps derived from escherichia coli , while cons and gbs infections are , at least partly , believed to be mediated by tlr2 . interestingly , the role of tlrs in nonbacterial - induced brain injury has also recently been highlighted . tlrs signal through the recruitment of intracellular adaptor proteins , followed by activation of protein kinases and transcription factors that induce the production of inflammatory mediators ( figure 1 ) . the adaptor protein myd88 is used by most tlrs , except tlr3 , while the trif adaptor protein is used only by tlr3 and tlr4 . lps - induced activation of tlr4 elicits , via both myd88 and trif , a broad inflammatory response in tissues , including the immature brain . there is relatively little information regarding the expression of tlrs in the developing brain . during embryonic life , protein expression of both tlr-3 and -8 has been identified [ 53 , 54 ] , while tlr-2 expression is relatively low before birth and increases during the first two weeks of life . we have shown that mrna for tlr1 - 9 is expressed in the neonatal mouse brain . it appears that some of the tlrs may play important roles during normal brain development , as tlr2 inhibits neural progenitor cell proliferation during the embryonic period , and tlr3 deficiency increases proliferation of neural progenitor cells , while tlr8 stimulation inhibits neurite outgrowth [ 5355 ] . in support , we , and others , have shown that systemic administration of lps results in brain injury in both fetal and newborn animals [ 5860 ] . these injuries appear , both histologically and by mri analysis , to be very similar to those found in preterm infants . furthermore , it is now well established that pre - exposure to lps can increase the vulnerability of the immature brain to hypoxia - ischemia ( hi ) , in both rats [ 62 , 63 ] and mice . these effects are tlr4 and myd88 dependent [ 64 , 66 ] . in a recent study , it was also shown that a very low dose of lps , specifically increased the vulnerability of the immature white matter . low - dose lps ( 0.05 mg / kg ) sensitized hi injury in p2 rat pups by selectively reducing myelin basic protein expression and the number of oligodendrocytes while increasing neuroinflammation and blood - brain barrier damage in the white matter . rat pups subjected to lps / hi at p1 responded with weak cytokine response , while there was a prominent upregulation of cytokines in p12 pups subjected to the same insult . interestingly , il-1 was upregulated at both ages ; il-1 injections sensitize the newborn brain to excitotoxicity and repeated il-1 exposure during the neonatal period induces preterm like brain injury in mice . although it has clearly been demonstrated that lps can increase the vulnerability to hi , under certain circumstances lps can also induce tolerance to brain injury . we have shown that the time interval between lps exposure and the subsequent hi is imperative to the outcome [ 71 , 72 ] , where a 24 h interval seems to induce a tolerant state that makes the brain less vulnerable . this has been confirmed by others who have implicated several possible mechanisms , including upregulation of corticosterone , which is further supported by the fact that administration of dexamethasone prevents learning impairment following lps / hi in neonatal rats . furthermore , akt - mediated enos upregulation in neurons and vascular endothelial cells have been implicated in lps - induced preconditioning . the importance of the time interval between lps and other insults seems to be a generalized phenomenon . we have recently demonstrated in an in vitro model that conditioned medium from lps - activated microglia affects the antioxidant nrf2 system and cell survival in astrocytes in a time - dependent manner . lps - induced inflammation had dual , time - dependent , effects on the nrf2 system in that sustained activation ( 72 h ) of gsk3beta and p38 downregulated the nrf2 system , possibly via the activation of histone deacetylases , changes that were not observed with a 24 h ( tolerance ) interval [ 76 , 77 ] . these studies support our previous report demonstrating that reductions in antioxidants were more pronounced when hi was preceded by lps injection in 8-day rats 3 days prior to the hi insult . compared to tlr4 , much less as mentioned above , tlr2 , tlr3 , and tlr8 can affect normal brain development [ 5355 ] . activation of tlr2 in neonatal mice decreases volume of cerebral gray matter , white matter in the forebrain , and cerebellar molecular layer . further , we have recently demonstrated the expression of both tlr1 and tlr2 in the neonatal mouse brain following hi . in these studies , tlr2 deficiency resulted in reduced infarct volume after hi , while tlr-1-deficient mice were not protected . maternal viral immune activation is believed to increase the risk of psychiatric disorders such as schizophrenia in offspring , and in order to examine this relationship , several authors have investigated the vulnerability of the fetal brain to synthetic double - stranded rna , polyriboinosinic - polyribocytidilic acid ( poly i : c ) , a tlr3 agonist . maternal injection with poly i : c towards the end of gestation ( g15 ) causes sensorimotor gating deficits in the adult offspring in mice and increased sensitivity to the locomotor - stimulating effects of mk-801 . maternal poly i : c injection on gd9 , but not gd17 , significantly impaired sensorimotor gating and reduced prefrontal dopamine d1 receptors in adulthood , whereas prenatal immune activation in late gestation impaired working memory , potentiated the locomotor reaction to a nmda - receptor antagonist , and reduced hippocampal nmda - receptor subunit 1 expression . in particular , poly i : c injections early during rodent pregnancy affect structural brain development , such as a transient decrease of myelin basic protein in the neonatal offspring and cerebellar pathology . e. coli infections are common in preterm neonates , and considerable evidence suggests that e. coli - induced inflammation play a role in the development of white matter damage in preterm infants . there is much less data available concerning the importance of two other common neonatal pathogens , cons and gbs , in perinatal brain injury . furthermore , it is becoming clear that tlrs have important roles during development and may be involved in both pathogen - induced damage as well as so called sterile hi - induced inflammation . in order to better understand the underlying causes of perinatal brain injury , the interaction between common neonatal pathogens and tlrs in the newborn brain deserves further investigation .

surgery for an anal fistula may result in recurrence or impairment of continence . the ideal treatment for an anal fistula should be associated with low recurrence rates , minimal incontinence and good quality of life . because of the risk of a change in continence with conventional techniques , sphincter - preserving techniques for the management complex anal fistulae have been evaluated . first , the anal fistula plug is made of lyophilized porcine intestinal submucosa . the anal fistula plug is expected to provide a collagen scaffold to promote tissue in growth and fistula healing . another addition to the sphincter - preserving options is the ligation of intersphincteric fistula tract procedure . this technique is based on the concept of secure closure of the internal opening and concomitant removal of infected cryptoglandular tissue in the intersphincteric plane . recently , cell therapy for an anal fistula has been described . adipose - derived stem cells have two biologic properties , namely , ability to suppress inflammation and differentiation potential . these properties are useful for the regeneration or the repair of damaged tissues . this article discusses the rationales for , the estimated efficacies of , and the limitations of new sphincter - preserving techniques for the treatment of anal fistulae . a growing body of evidence demonstrates that susceptibility and progression of both acute and chronic central nervous system disease in the newborn is closely associated with an innate immune response that can manifest from either direct infection and/or infection - triggered damage . a common feature of many of these diseases is the systemic exposure of the neonate to bacterial infections that elicit brain inflammation . in recent years , the importance of innate immune receptors in newborn brain injury , the so - called toll - like receptors , has been demonstrated . in this paper we will discuss how neonatal sepsis , with particular emphasis on escherichia coli , coagulase - negative staphylococci , and group b streptococcal infections in preterm infants , and toll - like receptor - mediated inflammation can increase the vulnerability of the newborn brain to injury .

assessing the degree of walking independence among inpatients is one of the important roles of physical therapists . overestimation of a patient s ability to walk can result in falls , whereas underestimation can lead to disuse syndrome due to a decrease in physical activity . preliminary research investigating cutoff values for parameters of walking independence in patients with stroke has reported the efficacy of physical performance examinations such as assessments of walking speed1 . however , preliminary studies often exclude stroke patients with cognitive disorders in order to increase the rate of identifying patients with walking independence and improve the reliability of examinations . stroke patients with cognitive disorders are often examined in the clinical setting2 , 3 , where the cutoff value for excluding stroke patients with cognitive disorders in preliminary research can not be used to evaluate those with cognitive disorders . as a result , walking independence in stroke patients with cognitive disorders is presently being assessed on the basis of subjective assessment by a physical therapist , and no clear protocol exists regarding this issue . therefore , determination of the cutoff values for parameters of walking independence in patients with stroke and cognitive disorders is necessary . but there have been few studies so far that have evaluated the reference values associated with walking independence in stroke patients with a cognitive impairment4 . the purpose of this study was to determine the threshold for classifying walking independence in stroke patients with and without cognitive disorders . the subjects included 130 patients with initial stroke hemiplegia admitted to the rehabilitation unit at koseinenkin kochi rehabilitation hospital in japan from april 2007 through december 2012 . patients were eligible for inclusion if they met the following criteria : ( 1 ) age 40 years ; ( 2 ) more than one month had passed since stroke onset ; ( 3 ) ability to complete the mini - mental state examination ( mmse ) ( total score : > 1)5 ; ( 4 ) first stroke , i.e. , no previous history of stroke ; ( 5 ) ability to walk a minimum of 10 m using mobility aids such as a cane or a brace ; and ( 6 ) a history of independent activities of daily living before the onset of stroke . we investigated basic information in addition to walking status , physical function , and performance of the patients during physical therapy based on their medical records . the survey items included gender , age , side of paralysis , functional independence measure ( fim ) for walking6 , mmse score , lower limb brunnstrom stage7 , one - leg standing time on the paralytic side , one - leg standing time on the non - paralytic side , and 10-m walking speed . for measuring the one - leg standing time , we measured the duration for which the patients were able to maintain one leg in the air without touching the floor8 , 9 . we excluded the measurements of one - leg standing time in the following cases : swaying while balancing , using the arms for balancing , hopping to maintain balance , and putting the foot down on the floor . the maximum measurement time was 60 s. the time before falling over was measured , and the maximum time of two repetitions was determined . the 10-m walking speed was used to quantify the ambulatory status in terms of a fast gait speed10 . the time from the starting line to a line marking 10 m was measured using a stopwatch . the participants performed two trials at each speed , and the calculated walking speed ( m / min ) was recorded . we conducted the statistical analysis according to the presence or absence of cognitive disorders . we classified the patients with mmse scores of 24 points into the high - score group and those with mmse scores of 23 points into the low - score group11 . the mmse measures various domains of cognitive functioning including memory , orientation to place and time , naming , reading , visuospatial orientation / construction ability , writing , and the ability to follow a 3-stage command . it is difficult to diagnose inattention and accomplishment dysfunction in patients with stroke using the mmse . therefore , patients with a deteriorated cognitive function may have been included in the high - score group . the statistical analysis was conducted to investigate the main factors associated with walking independence using a logistic regression analysis . the cutoff levels for the main factors used to distinguish walking independence were determined according to the compatibility of the regression models using the area under the curve ( auc ) of the receiver operating characteristic ( roc ) curve and the youden index12 . we calculated test diagnostics ( sensitivity , specificity , positive predictive value , negative predictive value , positive likelihood ratio , and negative likelihood ratio ) for a series of cutoff points . subjects were included after obtaining informed consent , and the study protocol was approved by the ethics committee of seirei christopher university . the high - score group included 69 subjects ( 53.1% ) , and the low - score group included 61 subjects ( 46.9% ) ( table 1table 1.characteristics of the high - score group and low - score groupvariablewalking statusdependentindependenthigh - score groupno . of patients2940age ( years)72.4 ( 11.8 ) 68.9 ( 11.3)female ( % ) 62.140.0left - sided hemiplegia ( % ) 58.652.5time after stroke onset to measurement ( m)3.5 ( 2.4)2.4 ( 2.2)brunnstrom stage ( 16)0.0.11.3.10.50.0.0.2.5.33*one - leg standing time on the paralytic side(s)0.4 ( 1.3)10.4 ( 15.4)*one - leg standing time on the non - paralytic side(s)2.3 ( 5.3)13.6 ( 18.2)*10-m walking speed ( m / min)23.9 ( 21.5)82.2 ( 47.5)*low - score groupno . of patients4021age ( years)79.4 ( 6.9)76.4 ( 7.9)female ( % ) 62.533.3*left - sided hemiplegia ( % ) 50.019.0*time after stroke onset to measurement ( m)3.1 ( 0.9)2.3 ( 0.7)*brunnstrom stage ( 16)0.0.3.6.16.150.0.0.1.1.19*one - leg standing time on the paralytic side(s)0.38 ( 0.74)3.8 ( 3.5)*one - leg standing time on the non - paralytic side(s)0.9 ( 1.7)4.8 ( 3.3)*10-m walking speed ( m / min)24.5 ( 17.6)71.7 ( 21.0)**p<0.05 the incidence of walking independence was 40 of 69 ( 58.0% ) patients with maintained cognitive function . according to the logistic regression analysis , the main factor associated with walking independence in the high - score group was the 10-m walking speed ( odds ratio , 1.076 ; 95% confidence interval , 1.0421.111 ) ( table 2table 2.results of the logistic regression analysisvariablebodds ratio95% confidence intervalhigh - score group10-m walking speed*0.0731.076 1.0421.111constant3.044identification rate87.0low - score group10-m walking speed*0.1071.1131.0451.186one - leg standing time on the non - paralytic side0.4951.6400.9692.777constant7.146identification rate88.5dependent variable : walking status ( dependent , 0 ; independent , 1 ) independent variable : 10-m walking speed and one - leg standing time on the non - paralytic side . odds ratio for a 1-second increase ) . as for the compatibility of the regression models of the roc curve , the auc was 0.921 . using a cutoff level for the 10-m walking speed of 41.4 m / min resulted in a sensitivity of 87.5% , specificity of 86.2% , positive predictive value of 90.0% , negative predictive value of 83.3% , positive likelihood ratio of 6.3 , and negative likelihood ratio of 0.2 ( fig . 1 . the auc was 0.921 in the high - score group . using a cutoff level for the 10-m walking speed of 41.4 the auc was 0.953 in the low - score group . using a cutoff level for the 10-m walking speed of 48.0 m / min resulted in a sensitivity of 95.2% and a specificity of 87.5% . ) . mann - whitney test dependent variable : walking status ( dependent , 0 ; independent , 1 ) independent variable : 10-m walking speed and one - leg standing time on the non - paralytic side . * odds ratio for a 1 m / min increase . odds ratio for a 1-second increase the auc was 0.921 in the high - score group . using a cutoff level for the 10-m walking speed of 41.4 the auc was 0.953 in the low - score group . using a cutoff level for the 10-m walking speed of 48.0 m / min resulted in a sensitivity of 95.2% and a specificity of 87.5% . the incidence of walking independence in the low - score group was 21 of 61 ( 34.4% ) patients with impaired cognitive function . according to the logistic regression analysis , the main factor associated with walking independence in the low - score group was the 10-m walking speed ( odds ratio , 1.113 ; 95% confidence interval , 1.0451.186 ) ( table 2 ) . with regard to the compatibility of the regression models of the roc curve , the auc was 0.953 . using a cutoff level for the 10-m walking speed of 48.0 m / min resulted in a sensitivity of 95.2% , specificity of 87.5% , positive predictive value of 89.5% , negative predictive value of 80.6% , positive likelihood ratio of 7.6 , and negative likelihood ratio of 0.1 ( fig . the patients in the low - score group based on the examinations for the presence of cognitive disorders accounted for 50% of all subjects . the subjects of this study included patients with initial stroke hemiplegia who had been treated in the rehabilitation unit at koseinenkin kochi rehabilitation hospital . on the basis of preliminary research it has become clear that patients with a history of several strokes can not be evaluated using the cutoff values employed in preliminary research that excludes patients with cognitive disorders . the results of this study showed that the primary factor associated with walking independence in the high - score group of stroke patients was the 10-m walking time . preliminary studies have found that the walking speed can be used to classify patients according to the degree of walking independence in the community15 . investigated the cutoff value of walking speed for community walkers with stroke and reported a positive predictive value of 93% and a negative predictive value of 57% for a cutoff value of 0.66 m / s , with an auc of 0.8515 . it is clear from these results that there is a standard value for classifying walking independence with walking speed . this study demonstrated the 10-m walking speed to be an effective parameter for identifying walking independence in the hospital . in the present study , the main factor associated with walking independence in the low - score group of stroke patients was the 10-m walking time . therefore , the 10-m walking speed is an effective parameter for classifying stroke patients with cognitive disorders according to the degree of walking independence in the hospital . examinations using complicated instructions decrease the reliability of assessments of physical function in patients with deteriorated cognitive function . it is therefore necessary to use simple examinations in patients with cognitive function disorders . we used the 10-m maximal walking speed to examine the walking independence status in this study . cognitive impairment in patients with stroke results in significant inattention and accomplishment dysfunction16 . patients with cognitive function disorders have difficulty in maintaining attention during long examinations and performing the required movements in assessments with complicated instructions17 . measuring the 10-m walking speed is an effective laboratory procedure due to its simplicity and allows for the determination of walking independence in patients evaluated with the mmse . data were obtained through an internet survey of u.s . adults self - identified as taking insulin to treat type 1 or type 2 diabetes ; the survey was conducted 13 june to 7 july 2008 by harris interactive , a contract research organization . patients were recruited by email if they had diabetes and currently used a syringe or insulin pen to deliver insulin . institutional review board approval for the study protocol was obtained from the human subject research committee of loyola university maryland . data collected from participants included the following : 1 ) basic demographic information ; 2 ) disease type , duration , complications , and treatment ; 3 ) perceived burden of insulin injections ; 4 ) the experience of injections ; 5 ) negative affect toward insulin injections ; and 6 ) frequency of skipping insulin injections . respondents reported whether they had ever been diagnosed with type 1 or type 2 diabetes , depression , obesity , or cardiovascular disease ( high blood pressure or heart disease ) and whether they treat their diabetes with diet , exercise , and medications . interference with eating and exercise was measured as the mean of two items asking , how much does the way you inject insulin interfere with eating / exercising when you want ? ( response options : 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal ) . interference with activities of daily living was measured as a count of the affirmative responses to the question , do your insulin injections have a negative effect on : social activities , recreational activities , sexual activity , work / career , family care - giving ? another measure of interference was whether the respondent plans daily activities around insulin injections ( 1 = yes , 0 = no ) . there were five measures dissatisfaction with injection time needed , ease of use , pain , inflammation / bruising , and embarrassment each measured by a single item ( response options : 1 = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied ) . i dread insulin injections ; injecting myself with insulin is the hardest part of managing my diabetes ; i have to mentally prepare myself before each injection ( response options : 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree ) . worry about hypoglycemia was measured by a single item ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . the dependent variable in this study was the response to the question , how often do you skip insulin injections that you know you should take ? ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . multiple regression analysis was used to assess independent relationships with frequency of skipping insulin injections . control variables ( demographic and disease characteristics ) were entered first , and then injection - related experience and attitudes were entered using stepwise criteria ( p < 0.05 ) . separate analyses were performed in the type 1 and type 2 diabetic populations to see whether associations differed between populations . data collected from participants included the following : 1 ) basic demographic information ; 2 ) disease type , duration , complications , and treatment ; 3 ) perceived burden of insulin injections ; 4 ) the experience of injections ; 5 ) negative affect toward insulin injections ; and 6 ) frequency of skipping insulin injections . respondents reported whether they had ever been diagnosed with type 1 or type 2 diabetes , depression , obesity , or cardiovascular disease ( high blood pressure or heart disease ) and whether they treat their diabetes with diet , exercise , and medications . interference with eating and exercise was measured as the mean of two items asking , how much does the way you inject insulin interfere with eating / exercising when you want ? ( response options : 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal ) . interference with activities of daily living was measured as a count of the affirmative responses to the question , do your insulin injections have a negative effect on : social activities , recreational activities , sexual activity , work / career , family care - giving ? ( possible range = 05 ) . another measure of interference was whether the respondent plans daily activities around insulin injections ( 1 = yes , 0 = no ) . there were five measures dissatisfaction with injection time needed , ease of use , pain , inflammation / bruising , and embarrassment each measured by a single item ( response options : 1 = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied ) . i dread insulin injections ; injecting myself with insulin is the hardest part of managing my diabetes ; i have to mentally prepare myself before each injection ( response options : 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree ) . worry about hypoglycemia was measured by a single item ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . the dependent variable in this study was the response to the question , how often do you skip insulin injections that you know you should take ? ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . interference with eating and exercise was measured as the mean of two items asking , how much does the way you inject insulin interfere with eating / exercising when you want ? ( response options : 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal ) . interference with activities of daily living was measured as a count of the affirmative responses to the question , do your insulin injections have a negative effect on : social activities , recreational activities , sexual activity , work / career , family care - giving ? another measure of interference was whether the respondent plans daily activities around insulin injections ( 1 = yes , 0 = no ) . there were five measures dissatisfaction with injection time needed , ease of use , pain , inflammation / bruising , and embarrassment each measured by a single item ( response options : 1 = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied ) . i dread insulin injections ; injecting myself with insulin is the hardest part of managing my diabetes ; i have to mentally prepare myself before each injection ( response options : 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree ) . worry about hypoglycemia was measured by a single item ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . the dependent variable in this study was the response to the question , how often do you skip insulin injections that you know you should take ? ( response options : 1 = never , 2 = rarely , 3 = sometimes , 4 = often ) . multiple regression analysis was used to assess independent relationships with frequency of skipping insulin injections . control variables ( demographic and disease characteristics ) were entered first , and then injection - related experience and attitudes were entered using stepwise criteria ( p < 0.05 ) . separate analyses were performed in the type 1 and type 2 diabetic populations to see whether associations differed between populations . the sample ( n = 502 ) was 55% male , 73% white , 11% hispanic , 11% african american , and 5% other race / ethnicity , with a mean age of 55 years ( table 1 ) . only about one - third ( 38% ) were presently employed , and those who were not employed included 8% students and 8% disabled ; the remainder were mostly retired or nonworking spouses . * 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal . = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied . 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree . 1 = never , 2 = rarely , 3 = sometimes , 4 = often . approximately one - third ( 32% ) reported having been diagnosed by a health care professional as having depression . a total of 77% of the sample said they had type 2 diabetes , and the rest said they had type 1 diabetes ; patients reported having diabetes for an average of almost 15 years . a total of 61% of the patient sample identified a primary care physician as their primary diabetes health care provider , whereas 28% named an endocrinologist and 11% named another ( nonphysician ) health care provider . of the sample , 39% reported engaging in physical activity and 55% said they followed a healthy diet . a total of 70% of patients surveyed said they took insulin using a syringe and 30% said they used a pen ; most ( 56% ) changed their needle with each injection . patients reported taking an average of 2.7 injections a day ( maximum of five recorded ) . a substantial minority of respondents ( 22% ) said they planned their daily activities around their insulin injections , and similar proportions reported that insulin injections interfered with their lives : 23% said insulin injections interfered with their eating / exercising schedule more than a little , and 25% said that insulin injections had a negative effect on one or more activity of daily living . further , a substantial minority of respondents ( 22% ) reported they had to mentally prepare themselves before each injection , and 33% identified they had some level of dread associated with taking their daily injections . attitudinal measures tended to fall below the halfway point of the response options ( i.e. , < 2.5 ) . respondents reported moderate levels of satisfaction with the pain and the inflammation and bruising associated with insulin injections ( the scores for pain and inflammation / bruising were significantly higher than those for embarrassment , time needed , and ease of use , p < 0.001 ) . a quarter ( 24% ) of respondents had a score representing negative affect toward injections ( they scored above the midpoint on the scale ) , and 21% reported often worrying about hypoglycemia . over half ( 57% ) of respondents reported skipping insulin injections they knew they should take ; 20% report skipping them sometimes or often . table 2 shows the results of the regression analysis of intentional insulin omission frequency . control variables ( demographic and diabetes characteristics ) accounted for 26% of the variance in intentional insulin omission . older respondents , those who were disabled , those with higher household income , and those who followed a healthy diet were significantly less likely to skip injections , whereas subjects who were students and had the highest education or type 2 diabetes and subjects who took more injections were significantly more likely to skip injections . multiple regression analysis of frequency of insulin injection omission * reference category = male . four measures of injection burden and experience had significant independent associations with higher levels of intentional insulin omission and accounted for an additional 10% of the variance . these included two aspects of interference ( planning one 's activities around insulin injections and injections interfering with activities of daily living ) and two aspects of the injection process itself ( pain and embarrassment ) . respondents who planned daily activities around insulin injections , those who said that taking injections interfered with activities of daily living , and those who reported injection - related pain or embarrassment intentionally skipped insulin injections more often . separate analyses using the variables listed in table 2 were performed among subjects with type 1 diabetes and subjects with type 2 diabetes ( results not shown ) . because there were more participants with type 2 diabetes , the overall model most closely resembled that for type 2 diabetes . only two variables significant in the overall model were not significant in the type 2 model being a student and following a healthy diet . only three variables were significant in the type 1 model following a healthy diet , number of daily insulin injections , and interference with activities of daily living ( being a student had a p value of 0.056 ) . the sample ( n = 502 ) was 55% male , 73% white , 11% hispanic , 11% african american , and 5% other race / ethnicity , with a mean age of 55 years ( table 1 ) . only about one - third ( 38% ) were presently employed , and those who were not employed included 8% students and 8% disabled ; the remainder were mostly retired or nonworking spouses . * 1 = not at all , 2 = a little , 3 = a moderate amount , 4 = a great deal . = very satisfied , 2 = satisfied , 3 = somewhat satisfied , 4 = not at all satisfied . 1 = strongly disagree , 2 = somewhat disagree , 3 = somewhat agree , 4 = strongly agree . 1 = never , 2 = rarely , 3 = sometimes , 4 = often . approximately one - third ( 32% ) reported having been diagnosed by a health care professional as having depression . a total of 77% of the sample said they had type 2 diabetes , and the rest said they had type 1 diabetes ; patients reported having diabetes for an average of almost 15 years . a total of 61% of the patient sample identified a primary care physician as their primary diabetes health care provider , whereas 28% named an endocrinologist and 11% named another ( nonphysician ) health care provider . of the sample , 39% reported engaging in physical activity and 55% said they followed a healthy diet . a total of 70% of patients surveyed said they took insulin using a syringe and 30% said they used a pen ; most ( 56% ) changed their needle with each injection . patients reported taking an average of 2.7 injections a day ( maximum of five recorded ) . a substantial minority of respondents ( 22% ) said they planned their daily activities around their insulin injections , and similar proportions reported that insulin injections interfered with their lives : 23% said insulin injections interfered with their eating / exercising schedule more than a little , and 25% said that insulin injections had a negative effect on one or more activity of daily living . further , a substantial minority of respondents ( 22% ) reported they had to mentally prepare themselves before each injection , and 33% identified they had some level of dread associated with taking their daily injections . attitudinal measures tended to fall below the halfway point of the response options ( i.e. , < 2.5 ) . respondents reported moderate levels of satisfaction with the pain and the inflammation and bruising associated with insulin injections ( the scores for pain and inflammation / bruising were significantly higher than those for embarrassment , time needed , and ease of use , p < 0.001 ) . a quarter ( 24% ) of respondents had a score representing negative affect toward injections ( they scored above the midpoint on the scale ) , and 21% reported often worrying about hypoglycemia . over half ( 57% ) of respondents reported skipping insulin injections they knew they should take ; 20% report skipping them sometimes or often . table 2 shows the results of the regression analysis of intentional insulin omission frequency . control variables ( demographic and diabetes characteristics ) accounted for 26% of the variance in intentional insulin omission . older respondents , those who were disabled , those with higher household income , and those who followed a healthy diet were significantly less likely to skip injections , whereas subjects who were students and had the highest education or type 2 diabetes and subjects who took more injections were significantly more likely to skip injections . multiple regression analysis of frequency of insulin injection omission * reference category = male . reference category = nonwhite . four measures of injection burden and experience had significant independent associations with higher levels of intentional insulin omission and accounted for an additional 10% of the variance . these included two aspects of interference ( planning one 's activities around insulin injections and injections interfering with activities of daily living ) and two aspects of the injection process itself ( pain and embarrassment ) . respondents who planned daily activities around insulin injections , those who said that taking injections interfered with activities of daily living , and those who reported injection - related pain or embarrassment intentionally skipped insulin injections more often . separate analyses using the variables listed in table 2 were performed among subjects with type 1 diabetes and subjects with type 2 diabetes ( results not shown ) . because there were more participants with type 2 diabetes , the overall model most closely resembled that for type 2 diabetes . only two variables significant in the overall model were not significant in the type 2 model being a student and following a healthy diet . only three variables were significant in the type 1 model following a healthy diet , number of daily insulin injections , and interference with activities of daily living ( being a student had a p value of 0.056 ) . this study suggests that intentional omission of insulin injections that should be taken occurs in the majority of adults using insulin to treat their diabetes and is common in 20% of these individuals . it also is associated with indicators of perceived burden and the experience of injections as painful and embarrassing . we found that respondents with higher household income , but not individuals with more education , were less likely to skip insulin injections they knew they should take . this may reflect easier access to medications and supplies among individuals with higher income , but it is also likely that higher socioeconomic status is associated with more access to diabetes education , higher health literacy , greater control over one 's daily routines , and better problem - solving skills ( 19 ) . our study appears to be among the first to identify an association between socioeconomic status and insulin omission . future research should seek to identify potential mediators of this relationship , i.e. , what links lower socioeconomic status to insulin omission . contrary to earlier reports ( 3,4 ) , we found no racial / ethnic differences in intentional insulin omission . this may be because we did not have enough nonwhite respondents to examine the different racial / ethnic groups separately . alternatively , this may be due to our controlling for income and education in the analysis , thereby eliminating the confounding of race / ethnicity with socioeconomic status . much prior research has suggested that intentional insulin omission is common among female adolescents with type 1 diabetes , serving as a weight control strategy and sometimes linked to eating disorders ( 10 ) . we found that students ( who were younger than nonstudents ) were more likely to skip injections they knew they should take , but this behavior was not more common among women than it was among men . we found no overall association between age and intentional insulin injection omission among patients with type 1 diabetes , suggesting that patients with type 1 diabetes age - out of this behavior by early adulthood , when they complete their education . ascertaining the validity of this interpretation would require following youth with type 1 diabetes as they age into adulthood to determine change in rate of insulin omission . our finding that , among individuals with type 2 diabetes , older respondents were less likely to skip insulin injections is consistent with earlier studies ( 3,4 ) . this suggests that there are parallel aging - out processes among individuals with type 1 and type 2 diabetes , but in type 2 diabetes , this process takes place later in the life course ( almost all people with type 2 diabetes are diagnosed as adults ) . ascertaining the validity of this interpretation would require following adults with type 2 diabetes as they age to determine change in rate of insulin omission . having type 2 diabetes was itself associated with higher levels of intentional omission of insulin injections . the beta for this variable ( 0.226 ) was approximately twice the size of the unadjusted eta ( 0.095 ) , reflecting the fact that controlling for confounding factors ( such as age and number of daily injections ) revealed a stronger underlying association . the independent association of type 2 diabetes with increased insulin omission may reflect the fact that patients with type 2 diabetes have a residual insulin response , reducing the immediate consequences of omitting an injection . thus , these individuals may feel less vulnerable to the effects of skipping insulin injections they know they should take . interestingly , whereas duration of diabetes was associated with the frequency of insulin injection omission , regression analysis revealed that duration of diabetes did not make an independent contribution to this behavior . that is , although insulin omission may be less common among individuals with longer duration of diabetes , this is likely a function of other factors such as age and type of diabetes rather than of duration per se . surprisingly , history of depression was not associated with insulin omission ; this contradicts findings from studies of general adherence ( 9,20 ) and of insulin omission among adolescents ( 10 ) . however , because current depression was not assessed , any concurrent association was likely to be lost . others have shown that depression symptom scores fluctuate substantially over relatively short periods of time ; individuals with elevated depression symptoms at a given point in time are likely to not report elevated symptoms 6 months later ( 21,22 ) . in addition , depression symptoms across the whole range of severity symptoms have been shown to predict regimen adherence more powerfully than diagnosed depression ( 23 ) . being disabled was associated with less insulin omission ; this may be due to a variety of reasons , including their receiving more assistance with care , or making a greater effort to compensate for poor health . two aspects of patients ' treatment regimens were associated with increased insulin omission respondents who took more injections each day and those who did not follow a healthy diet were more likely to skip injections . that dietary nonadherence is associated with insulin nonadherence more frequent injection omission among individuals taking more injections could reflect the frequently reported finding that more complex regimens are associated with lower levels of adherence ( 6 ) . it might also be that the impact of skipping a shot is reduced among individuals who take more shots . our study suggests that insulin omission is affected by the perceived burden of insulin therapy ( i.e. , having to plan one 's life around insulin injections and feeling that the insulin regimen interferes with activities of daily living such as social activities , work - related activities , and family care - giving responsibilities ) . we offer one caveat regarding our findings ; we do not believe that the behavior of planning one 's day around insulin injections actually increases the level of insulin injection omission , but we do believe that feeling that one has to plan around one 's injections is associated with higher frequency of skipping insulin injections one should take . that is , when there is a conflict between scheduling of treatment and life activities , one can either plan one 's activities in a way that reduces this conflict or deal with the conflict by ignoring treatment needs . reducing the perceived burden of insulin injections may require more effort from health care providers . as we have suggested elsewhere , providers must find out what the specific issues are for each patient and work with that patient to develop solutions that will work for him or her ( 24 ) . we note that the measure of interference with eating and exercise was significantly associated with insulin injection omission until interference with activities of daily living was entered into the model ( results not shown ) . thus , while interference with eating and exercise might be part of the burden of insulin therapy , interference with other aspects of daily living had a more substantial association with insulin omission . our study suggests that insulin omission may be affected by the immediate experience of injecting insulin as painful and embarrassing ( but not dissatisfaction with time needed , ease of use , or skin inflammation / bruising ) . there are numerous device - related strategies for reducing pain and embarrassment , including insulin pens , finer gauge needles , injection ports , needleless injectors , and other injection assistance devices . however , we have found that patients do not feel that their health care providers are giving them adequate assistance in managing these problems , even when they raise the issue with their providers ( 18 ) . we note that the measure of negative affect toward injections was significantly associated with insulin omission until dissatisfaction with injection embarrassment and pain were entered into the model ( results not shown ) . this suggests that addressing pain and embarrassment may reduce not only insulin omission , but also the emotional burden of injections , thereby enhancing psychological well - being . it is interesting that worry about hypoglycemia did not predict intentional omission of insulin injections , even though worry about hypoglycemia was high in the study population . this suggests that patients may address this worry by eating more or lowering insulin doses rather than by skipping injections altogether . strengths of the study include the large sample of diabetic patients drawn from a general population and the fact that the sample was weighted to be nationally representative . however , patients volunteer for the panel from which respondents were drawn and may not be representative of all patients ( e.g. , they may be more adherent with their treatment regimens ) . limitations of the study include the fact that there was no objective measure of insulin use ( e.g. , pharmacy records ) . moreover , while our measure of insulin omission was very specific ( i.e. , skipping injections that respondents knew they should take ) , it is possible that some respondents included injections they did not skip intentionally , but rather simply forgot to take . respondents might also have included scheduled injections that were appropriately skipped because a meal was not eaten or blood glucose levels were very low . this could explain ( at least in part ) the association between following a healthy diet and fewer skipped injections . more disciplined eating behavior reduces meal skipping , which is an often - cited reason for skipping insulin injections . this is consistent with the fact that the relationship between our diet measure and insulin omission is present only among individuals with type 1 diabetes where closer matching of food and insulin is required . finally , our study probably underestimated the level of insulin nonadherence in this population because it did not capture instances in which patients took an injection but gave less than the amount of insulin they knew was needed for optimal glucose control . our findings make clear that while most patients did not report regular omission of insulin injections , a substantial number did . thus , our findings suggest that it is important to identify patients who omit insulin and to be aware of the potential risk factors identified here . although much attention has been focused on insulin omission among adolescents with type 1 diabetes , our findings suggest that among adults , individuals with type 2 diabetes are at higher risk . patients who are not adhering to other elements of the treatment regimen , especially diet , also may be at risk for insulin omission . for patients who report injection - related problems ( interference with daily activities , injection pain , and embarrassment ) , providers should consider recommending strategies and tools for addressing these problems to prevent insulin omission . we found that respondents with higher household income , but not individuals with more education , were less likely to skip insulin injections they knew they should take . this may reflect easier access to medications and supplies among individuals with higher income , but it is also likely that higher socioeconomic status is associated with more access to diabetes education , higher health literacy , greater control over one 's daily routines , and better problem - solving skills ( 19 ) . our study appears to be among the first to identify an association between socioeconomic status and insulin omission . future research should seek to identify potential mediators of this relationship , i.e. , what links lower socioeconomic status to insulin omission . contrary to earlier reports ( 3,4 ) , we found no racial / ethnic differences in intentional insulin omission . this may be because we did not have enough nonwhite respondents to examine the different racial / ethnic groups separately . alternatively , this may be due to our controlling for income and education in the analysis , thereby eliminating the confounding of race / ethnicity with socioeconomic status . much prior research has suggested that intentional insulin omission is common among female adolescents with type 1 diabetes , serving as a weight control strategy and sometimes linked to eating disorders ( 10 ) . we found that students ( who were younger than nonstudents ) were more likely to skip injections they knew they should take , but this behavior was not more common among women than it was among men . we found no overall association between age and intentional insulin injection omission among patients with type 1 diabetes , suggesting that patients with type 1 diabetes age - out of this behavior by early adulthood , when they complete their education . ascertaining the validity of this interpretation would require following youth with type 1 diabetes as they age into adulthood to determine change in rate of insulin omission . our finding that , among individuals with type 2 diabetes , older respondents were less likely to skip insulin injections is consistent with earlier studies ( 3,4 ) . this suggests that there are parallel aging - out processes among individuals with type 1 and type 2 diabetes , but in type 2 diabetes , this process takes place later in the life course ( almost all people with type 2 diabetes are diagnosed as adults ) . ascertaining the validity of this interpretation would require following adults with type 2 diabetes as they age to determine change in rate of insulin omission . having type 2 diabetes was itself associated with higher levels of intentional omission of insulin injections . the beta for this variable ( 0.226 ) was approximately twice the size of the unadjusted eta ( 0.095 ) , reflecting the fact that controlling for confounding factors ( such as age and number of daily injections ) revealed a stronger underlying association . the independent association of type 2 diabetes with increased insulin omission may reflect the fact that patients with type 2 diabetes have a residual insulin response , reducing the immediate consequences of omitting an injection . thus , these individuals may feel less vulnerable to the effects of skipping insulin injections they know they should take . interestingly , whereas duration of diabetes was associated with the frequency of insulin injection omission , regression analysis revealed that duration of diabetes did not make an independent contribution to this behavior . that is , although insulin omission may be less common among individuals with longer duration of diabetes , this is likely a function of other factors such as age and type of diabetes rather than of duration per se . surprisingly , history of depression was not associated with insulin omission ; this contradicts findings from studies of general adherence ( 9,20 ) and of insulin omission among adolescents ( 10 ) . however , because current depression was not assessed , any concurrent association was likely to be lost . others have shown that depression symptom scores fluctuate substantially over relatively short periods of time ; individuals with elevated depression symptoms at a given point in time are likely to not report elevated symptoms 6 months later ( 21,22 ) . in addition , depression symptoms across the whole range of severity symptoms have been shown to predict regimen adherence more powerfully than diagnosed depression ( 23 ) . being disabled was associated with less insulin omission ; this may be due to a variety of reasons , including their receiving more assistance with care , or making a greater effort to compensate for poor health . two aspects of patients ' treatment regimens were associated with increased insulin omission respondents who took more injections each day and those who did not follow a healthy diet were more likely to skip injections . that dietary nonadherence is associated with insulin nonadherence more frequent injection omission among individuals taking more injections could reflect the frequently reported finding that more complex regimens are associated with lower levels of adherence ( 6 ) . it might also be that the impact of skipping a shot is reduced among individuals who take more shots . our study suggests that insulin omission is affected by the perceived burden of insulin therapy ( i.e. , having to plan one 's life around insulin injections and feeling that the insulin regimen interferes with activities of daily living such as social activities , work - related activities , and family care - giving responsibilities ) . we offer one caveat regarding our findings ; we do not believe that the behavior of planning one 's day around insulin injections actually increases the level of insulin injection omission , but we do believe that feeling that one has to plan around one 's injections is associated with higher frequency of skipping insulin injections one should take . that is , when there is a conflict between scheduling of treatment and life activities , one can either plan one 's activities in a way that reduces this conflict or deal with the conflict by ignoring treatment needs . reducing the perceived burden of insulin injections may require more effort from health care providers . as we have suggested elsewhere , providers must find out what the specific issues are for each patient and work with that patient to develop solutions that will work for him or her ( 24 ) . we note that the measure of interference with eating and exercise was significantly associated with insulin injection omission until interference with activities of daily living was entered into the model ( results not shown ) . thus , while interference with eating and exercise might be part of the burden of insulin therapy , interference with other aspects of daily living had a more substantial association with insulin omission . our study suggests that insulin omission may be affected by the immediate experience of injecting insulin as painful and embarrassing ( but not dissatisfaction with time needed , ease of use , or skin inflammation / bruising ) . there are numerous device - related strategies for reducing pain and embarrassment , including insulin pens , finer gauge needles , injection ports , needleless injectors , and other injection assistance devices . however , we have found that patients do not feel that their health care providers are giving them adequate assistance in managing these problems , even when they raise the issue with their providers ( 18 ) . we note that the measure of negative affect toward injections was significantly associated with insulin omission until dissatisfaction with injection embarrassment and pain were entered into the model ( results not shown ) . this suggests that addressing pain and embarrassment may reduce not only insulin omission , but also the emotional burden of injections , thereby enhancing psychological well - being . it is interesting that worry about hypoglycemia did not predict intentional omission of insulin injections , even though worry about hypoglycemia was high in the study population . this suggests that patients may address this worry by eating more or lowering insulin doses rather than by skipping injections altogether . strengths of the study include the large sample of diabetic patients drawn from a general population and the fact that the sample was weighted to be nationally representative . however , patients volunteer for the panel from which respondents were drawn and may not be representative of all patients ( e.g. , they may be more adherent with their treatment regimens ) . limitations of the study include the fact that there was no objective measure of insulin use ( e.g. , pharmacy records ) . moreover , while our measure of insulin omission was very specific ( i.e. , skipping injections that respondents knew they should take ) , it is possible that some respondents included injections they did not skip intentionally , but rather simply forgot to take . respondents might also have included scheduled injections that were appropriately skipped because a meal was not eaten or blood glucose levels were very low . this could explain ( at least in part ) the association between following a healthy diet and fewer skipped injections . more disciplined eating behavior reduces meal skipping , which is an often - cited reason for skipping insulin injections . this is consistent with the fact that the relationship between our diet measure and insulin omission is present only among individuals with type 1 diabetes where closer matching of food and insulin is required . finally , our study probably underestimated the level of insulin nonadherence in this population because it did not capture instances in which patients took an injection but gave less than the amount of insulin they knew was needed for optimal glucose control . our findings make clear that while most patients did not report regular omission of insulin injections , a substantial number did . thus , our findings suggest that it is important to identify patients who omit insulin and to be aware of the potential risk factors identified here . although much attention has been focused on insulin omission among adolescents with type 1 diabetes , our findings suggest that among adults , individuals with type 2 diabetes are at higher risk . patients who are not adhering to other elements of the treatment regimen , especially diet , also may be at risk for insulin omission . for patients who report injection - related problems ( interference with daily activities , injection pain , and embarrassment ) , providers should consider recommending strategies and tools for addressing these problems to prevent insulin omission .

[ purpose ] the aim of this study was to determine the threshold for classifying walking independence in stroke patients with and without cognitive disorders . [ subjects ] the subjects were 130 patients with initial stroke hemiplegia . [ methods ] the following factors were analyzed for associations with walking independence : brunnstrom stage , one - leg standing time on the paralytic side , one - leg standing time on the non - paralytic side , and 10-m walking speed . we classified the patients with mini - mental state examination ( mmse ) scores 24 points into the high - score group and those with mmse scores of 23 points into the low - score group and examined the main factors and cutoff values associated with walking independence in each group . [ results ] the high - score group included 69 subjects ( 53.1% ) , and the low - score group included 61 subjects ( 46.9% ) . the primary factor associated with high mmse scores among the stroke patients was the 10-m walking time . using a cutoff level for the 10-m walking speed of 41.4 m / min resulted in a positive likelihood ratio of 6.3 . the primary factor associated with low mmse scores among the stroke patients was the 10-m walking time . using a cutoff level for the 10-m walking speed of 48.0 m / min resulted in a positive likelihood ratio of 7.6 . [ conclusion ] the cutoff value for the 10-m walking speed can be used to evaluate walking independence in patients with stroke among patients with high or low mmse scores . objectivethe purpose of this study was to assess factors associated with patient frequency of intentionally skipping insulin injections.research design and methodsdata were obtained through an internet survey of 502 u.s . adults self - identified as taking insulin by injection to treat type 1 or type 2 diabetes . multiple regression analysis assessed independent associations of various demographic , disease , and injection - specific factors with insulin omission.resultsintentional insulin omission was reported by more than half of respondents ; regular omission was reported by 20% . significant independent risk factors for insulin omission were younger age , lower income and higher education , type 2 diabetes , not following a healthy diet , taking more daily injections , interference of injections with daily activities , and injection pain and embarrassment . risk factors differed between type 1 and type 2 diabetic patients , with diet nonadherence more prominent in type 1 diabetes and age , education , income , pain , and embarrassment more prominent in type 2 diabetes.conclusionswhereas most patients did not report regular intentional omission of insulin injections , a substantial number did . our findings suggest that it is important to identify patients who intentionally omit insulin and be aware of the potential risk factors identified here . for patients who report injection - related problems ( interference with daily activities , injection pain , and embarrassment ) , providers should consider recommending strategies and tools for addressing these problems to increase adherence to prescribed insulin regimens . this could improve clinical outcomes .

otosclerosis is a primary , hereditary , localized , metabolic bone disease of bone derived only from the bony otic capsule . it is characterized by alternating phases of abnormal removal of mature bone of the otic capsule by osteoclasts , and replacement with new woven bone of greater thickness , cellularity , and vascularity . because it has predilection for the fissula ante fenestrum , involvement of the stapedial footplate may result in its fixation and an asymmetric conductive hearing loss ( stapedial otosclerosis ) whereby sounds partly fail to reach the inner ear ( cochlea ) [ 1 , 2 ] . involvement of other parts of the otic capsule ( cochlear otosclerosis ) may result more often in cochlear symptoms ( especially a high - frequency sensorineural hearing loss , which usually manifests late in the disease , and 75% tinnitus ) and vestibular symptoms ( in 25% of cases , most commonly a sense of disequilibrium , occasionally attacks of positional and motion - related vertigo with rotatory nystagmus ) , or a combination of these 2 symptoms ( combined otosclerosis with mixed hearing loss ) . diagnosis of otosclerosis is usually based on family history and clinical and audiologic examination [ 46 ] . stapedectomy / stapedotomy is the surgery of choice ; placing a piston prosthesis into the vestibule through the oval window fenestra provides mobile continuity between the inner ear and ossicular chain . testing the vestibular function in patients with otosclerosis is of particular interest . the bithermal caloric test ( bct ) is representative physiological test of the function of the lateral semicircular canal ( lscc ) and its afferents , and vemps mainly reflects the function of the saccule and its afferents . it is an objective , quick , and non - invasive measure of saccule function that , along with the utricle , primarily respond to linear acceleration in any direction . the saccule , which is the lower of the 2 otolithic organs , is most sensitive to gravity because it is in the vertical plane . it also has a slight sound sensitivity , which is thought to be a remnant from the function of the saccule as an organ of hearing in lower animals . the acoustic sensitivity of the saccule has been attributed to its proximity to the footplate of the stapes , which leads to its mechanical stimulation during stapedial motion in response to sound and can be measured . this is the basis of the vemp test , which is almost entirely saccular in origin . it has been widely used since 1992 after colebatch and halmagy described it as it is known in modern days . they selected the scm as their standard recording site of vestibular and myogenic response to sound [ 911 ] . vemp testing utilizes sophisticated computerized equipment to interpret waveforms on a computer screen for each ear , each type of stimulus used , and each intensity level . the purpose of the air - conducted vemp is to determine if the saccule stimulated by the sound , the activated inferior vestibular nerve ( ivn ) , central connections in the brainstem [ lateral vestibular nucleus ( deiters s nucleus ) , accessory nerve nucleus ] , the 11 nerve , and the sternocleidomastoid muscle ( scm , mostly ipsilaterally ) via the medial vestibulospinal tract ( mvst ) are intact and working normally . bone - conducted vemp is a useful measure of vestibular function , especially in the presence of conductive hearing loss , such as in otosclerosis , where the bone - conducted sound activates the vestibular apparatus more effectively than air - conducted sound [ 1317 ] . the bone - conducted sound can stimulate the saccule and other parts of the vestibular end organs on both sides as the utricle . the objectives of the current study were to : ( a ) investigate the audio - vestibular profile of stapedectomy candidates by analyzing the audiogram , vemp , and calorics results ; ( b ) evaluate the cause of vestibular dysfunction in patients with otosclerosis ; and ( c ) to determine the clinical utility of vemps in the differential diagnosis of otosclerosis and otologic diseases that can cause pseudo - conductive hearing loss such as scds and lvas . seventy - four caucasian ( 51 female , 23 male ) patients were seen in our academic tertiary referral centre otolaryngology department of hippokration hospital , university of athens , and were prospectively assessed as candidates for stapes surgery for otosclerosis ( mean age 38.09 years , range 2360 years).we selected an age range of 2360 years because there is evidence that the vemp response rate decreases after age 60 [ 1820 ] . no patient had a positive history for chronic otitis media , skull trauma , superior canal dehiscence syndrome ( scds ) , or large vestibular aqueduct syndrome ( lvas ) . tinnitus was present in 62 ( 83.7% ) subjects , 24 ( 32.4% ) patients reported dizziness , with 1 case of left - sided bppv treated with epley manoeuvre , and another 2 presented with first - degree right - beating spontaneous nystagmus . all the patients enrolled in this study were evaluated prior to stapedectomy using a standard diagnostic protocol that included audiological assessment consisting of pure tone and speech audiometry , tympanometry with stapedial reflexes , eng with bithermal caloric testing ( bct ) , and vemp test . of these patients , 1 underwent high - resolution computed tomography ( hrct ) for further evaluation of the disease . in all patients , the mean of 4-frequency pure tone hearing thresholds ( 4-pta at 500 , 1000 , 2000 , and 4000 hz ) was measured for both air and bone conduction and consequently the abg ( air - bone gap ) . vemps were recorded with a gn otometrics ( taastrup , denmark ) ics chartr ep version 5.2 analyser with a 2-channel averaging capacity . vemps were recorded while patients were seated in an upright position and instructed to maintain their heads turned contralaterally to the stimulated ear to achieve intense and constant sternocleidomastoid muscular effort during the whole recording period of each trial .the skin was scrubbed , and the impedance of the recording electrodes was maintained below 5 kohms . the 2 active recording electrodes were placed symmetrically at the middle third of each scm , the reference electrode was placed on the upper forehead , and the ground electrode was placed at the middle of the forehead . the acoustic stimulus ( loud , short tone bursts ) intensity range was 95102 db hl for ac - vemps and 4265 db hl for bc - vemps [ 500 hz tone bursts were given at 95 db hl for ac - vemps and 42 db hl for bc - vemps ; 1000hz tone bursts were given at 102 db hl for ac - vemps and 65 db hl for bc - vemps ; rate 5.1/second , ramp 1 millisecond ( msec ) , plateau 0 ms ] . stimuli were delivered monaurally via headphones ( tdh-40 , telephonics , new york , usa ) for elicitation of vemps evoked by air - conducted signal ( ac - vemps ) and the bone oscillator b71 placed at the ipsilateral mastoid for evocation of responses by bone - conducted stimuli ( bc - vemps ) with no contralateral masking , and the myogenic potential was recorded ipsilaterally by surface electrodes . the electromyographic ( emg ) activity of the ipsilateral sternocleidomastoid muscle was recorded , and every trial of 150 stimuli was averaged and repeated twice to verify the reproducibility of the waveform , and to provide the final vestibular evoked myogenic potential waveform . the emg signal from each side was amplified and bandpass - filtered ( high - pass 2 hz , low - pass 500 hz ) . the peak latencies of the first positive - negative component of the vestibular evoked myogenic potential response ( p1 and n1 ) were measured for each patient . the vestibular evoked myogenic potential response was considered to be absent when there were no recognizable or reproducible biphasic waveforms , or when the amplitude of the potential was less than 20 mv . electronystagmography recordings ( eng ) were performed with a life - tech model 3002 electronystagmograph ( houston , texas , usa ) . a hortmann airmatic ( neurootometrie , gn otometrics , taastrup , denmark ) air irrigator any caloric asymmetry of more than 22% was defined as canal paresis . the statistical package for the social sciences version 11.0 software student s t - test , anova and chi square testing were used to compare different values . the criterion for statistical significance was set at a p - value of 0.05 ( 2-tailed ) . for comparison , 35 healthy volunteers adults ( 17 men and 18 women ; mean age 37.3 years)for ac - vemps and 13 healthy adults ( 6 men and 7 female ; mean age 37.76 years ) for bc - vemps , without previous hearing , vestibular , or neurological disorders , were also enrolled in this study . all volunteers had a normal otoscopic examination and a normal pure tone audiometric ( pta ) threshold . ac vestibular evoked myogenic potential waveforms were obtained on both sides in all of the 35 healthy volunteers ( 70 ears ) and bc vestibular evoked myogenic potential waveforms in all of the 13 healthy volunteers ( 26 ears ) . the mean ac p1 value was 16.26 ms ( sd 1.32 ) and the mean ac n1 value was 24.42 ( sd 2.52 ) . the mean bc p1 value was 14.09 ( sd 2.70 ) and the mean bc n1 value was 22.30 ( sd 3.91).the p1 and n1 delay was defined as any value greater than the mean plus 2 sds of the normal population ( i.e. , any p1 value greater than 18.49 ms and any n1 value greater than 30.12 ms ) was considered delayed . this study was conducted with the understanding and the consent of all subjects , patients , and volunteers before inclusion . pre - operatively , ac - vemp was performed in 126 ears and ac - vemp and bc - vemp were performed in 109 ears each . ac - vemp was present in 34 ears , while bc - vemp was recordable in 48 ears . the mean ac 4-pta , mean bc 4-pta , and mean abg ( air - bone gap ) were compared between the group of ears with ac - vemp presence and ac - vemp absence , respectively . the same was done between the group of ears with bc - vemp presence and bc - vemp absence . the results appear in table 1 . statistically significant differences ( student s t - test ) were found in all 3 parameters between the group with ac - vemp(+ ) ( present ) and ac - vemp( ) ( absent ) . in contrast , the comparisons between the ears with presence and absence of bc - vemp were not significant . table 2 shows the combination of the 2 tests ( ac - vemp and bc - vemp ) in a total of 109 ears . the response rates of present ac - vemps and present bc - vemps were 29.36% ( 32 ears ) and 44.03% ( 48 ears ) respectively . chi - square revealed a strong correlation between these 2 investigations ; when ac - vemp was present , it tended to be the bc - vemp ; but when ac - vemp was absent , so was the bc - vemp . the percentage of vemp presence in healthy subjects was shown to be about 100% in controls without history of ear disease or head trauma . table 3 shows no significant statistical difference between the means of the latencies p1 , ( nor of n1 ) of the normal ears and the ears affected with otosclerosis with recordable ac - vemp . on the contrary , a significant statistical difference was noted between the means of latencies p1(and n1 ) of normal ears and ears affected by otosclerosis with elicited bc - vemp . mean abg in each 1 of the 4 groups formed by the combination of ac and bc - vemp appears in table 4 . no significant effect of the factor group was found , meaning that no statistical differences existed among the abg means ( one - way between - subjects anova ) . on the contrary , anova showed statistically significant differences among the means of ac 4-pta between the group of ears with presence and absence of both air- and bone - conducted vemp . [ the mean of the group with both vemp present ( 48.55 db ) differs statistically from the mean of the group with both vemp absent ( 59.66 db ) ] . however , no statistical differences were found among the bc 4-pta in the same 4 groups . preoperatively , the results of caloric responses were assessed in relation with the results of ac and bc - vemp ( chi - square ) in 109 otosclerotic ears . table 5 shows no correlation either between canal paresis ( cp ) occurrence and air - conducted vemp presence ( p=0.461 ) , or with canal paresis and bone - conducted vemp presence ( p=0.366 ) . vestibular symptoms occur in approximately 2037% of patients affected by otosclerosis , but their pathogenesis remains unclear . in our study , 24 ( 32.4% ) of 74 patients presented with symptoms of dizziness in the form of vertigo or disequilibrium ( after carefully completing a medical questionnaire , only patients with symptoms compatible with labyrinthine origin were found in the dizziness group ) . to study the relationship between dizziness , means of ac 4-pta , bc 4-pta , ac - vemp , bc - vemp and calorics in patients affected by otosclerosis , pre - operatively , the following groups of patients were defined as : group d ( patients who complained of dizziness ) and group nd ( patients with no complaint of dizziness ) [ 3133 ] . the d group was composed of 42 ears and the nd group was composed of 84 otosclerotic ears . the mean of ac 4-pta thresholds in groups d and nd were 57.63(sd 16.81 ) and 53.22(sd 16.65 ) db , respectively ( table 6 ) . there were no significant differences between the 2 groups ( student s t test , p=0.167 ) . mean bc 4-pta thresholds in groups d and nd were 30.78(sd 9.88 ) and 27.65 ( sd 11.36 ) db , respectively . no significant difference was observed between the 2 groups ( student s t - test , p=0.131 ) ( table 6 ) . for caloric testing , 19 of 42 ( 45.2% ) ears showed abnormal results in d group with 11 unilateral cp ( 1 presented with left - sided bppv ) and 4 bilateral cp ( 1 presented with first - degree right - beating spontaneous nystagmus ) . in the nd group , 24 out of 84 ( 28.6% ) ears showed abnormal calorics , with 16 unilateral and 4 bilateral cp . no statistical relationship was found between the calorics results and the appearance of dizziness ( , p=0.06 ) ( table 6 ) . ( , p=0.06 for ac - vemp , p=0.598 for bc - vemp ) ( table 6 ) . to assess the possible relation of the factors of dizziness and caloric results in the measurements of ac 4-pta , bc 4-pta and abg , a 2-way anova was carried out with dizziness having 2 levels [ i.e. , yes ( dizziness present ) and no ( no dizziness ) ] and caloric results having 3 levels ( i.e. , cp at the ipsilateral ear [ the ear in which pta and air - bone gap(abg ) are measured and enter the study ] , cp at the contralateral ear ( so the ear measured has normal calorics ) , and normal calorics ( referring to cases with no canal paresis to either ear ) . for cases with bilateral cp in both ears were counted as ipsilateral cp . concerning ac and bc 4-pta , the anova did not show a significant main effect either for dizziness or for calorics , meaning that there were no significant differences of the means of the above measurements , neither between the dizzy and non - dizzy group , nor between the ears with cp and the ears with normal calorics . however , a statistically significant interaction was found between the 2 factors ( dizziness and calorics ) in relation to ac and bc - pta thresholds . in the dizziness group ( d ) , ears with cp showed much higher means of ac and bc 4-pta , whereas in the non dizziness ( nd ) group the contralateral ear ( with normal calorics ) showed higher ac and bc 4-pta means ( figures 1 and 2 ) . concerning the abg , no significances were found ( p=0.364 for the interactions , p=0.971 , p=0.522 for the main effects of dizziness and calorics , respectively ) . pre - operatively , ac - vemp was performed in 126 ears and ac - vemp and bc - vemp were performed in 109 ears each . ac - vemp was present in 34 ears , while bc - vemp was recordable in 48 ears . the mean ac 4-pta , mean bc 4-pta , and mean abg ( air - bone gap ) were compared between the group of ears with ac - vemp presence and ac - vemp absence , respectively . the same was done between the group of ears with bc - vemp presence and bc - vemp absence . the results appear in table 1 . statistically significant differences ( student s t - test ) were found in all 3 parameters between the group with ac - vemp(+ ) ( present ) and ac - vemp( ) ( absent ) . in contrast , the comparisons between the ears with presence and absence of bc - vemp were not significant . table 2 shows the combination of the 2 tests ( ac - vemp and bc - vemp ) in a total of 109 ears . the response rates of present ac - vemps and present bc - vemps were 29.36% ( 32 ears ) and 44.03% ( 48 ears ) respectively . chi - square revealed a strong correlation between these 2 investigations ; when ac - vemp was present , it tended to be the bc - vemp ; but when ac - vemp was absent , so was the bc - vemp . the percentage of vemp presence in healthy subjects was shown to be about 100% in controls without history of ear disease or head trauma . table 3 shows no significant statistical difference between the means of the latencies p1 , ( nor of n1 ) of the normal ears and the ears affected with otosclerosis with recordable ac - vemp . on the contrary , a significant statistical difference was noted between the means of latencies p1(and n1 ) of normal ears and ears affected by otosclerosis with elicited bc - vemp . mean abg in each 1 of the 4 groups formed by the combination of ac and bc - vemp appears in table 4 . no significant effect of the factor group was found , meaning that no statistical differences existed among the abg means ( one - way between - subjects anova ) . on the contrary , anova showed statistically significant differences among the means of ac 4-pta between the group of ears with presence and absence of both air- and bone - conducted vemp . [ the mean of the group with both vemp present ( 48.55 db ) differs statistically from the mean of the group with both vemp absent ( 59.66 db ) ] . however , no statistical differences were found among the bc 4-pta in the same 4 groups . preoperatively , the results of caloric responses were assessed in relation with the results of ac and bc - vemp ( chi - square ) in 109 otosclerotic ears . table 5 shows no correlation either between canal paresis ( cp ) occurrence and air - conducted vemp presence ( p=0.461 ) , or with canal paresis and bone - conducted vemp presence ( p=0.366 ) . vestibular symptoms occur in approximately 2037% of patients affected by otosclerosis , but their pathogenesis remains unclear . in our study , 24 ( 32.4% ) of 74 patients presented with symptoms of dizziness in the form of vertigo or disequilibrium ( after carefully completing a medical questionnaire , only patients with symptoms compatible with labyrinthine origin were found in the dizziness group ) . to study the relationship between dizziness , means of ac 4-pta , bc 4-pta , ac - vemp , bc - vemp and calorics in patients affected by otosclerosis , pre - operatively , the following groups of patients were defined as : group d ( patients who complained of dizziness ) and group nd ( patients with no complaint of dizziness ) [ 3133 ] . the d group was composed of 42 ears and the nd group was composed of 84 otosclerotic ears . the mean of ac 4-pta thresholds in groups d and nd were 57.63(sd 16.81 ) and 53.22(sd 16.65 ) db , respectively ( table 6 ) . there were no significant differences between the 2 groups ( student s t test , p=0.167 ) . mean bc 4-pta thresholds in groups d and nd were 30.78(sd 9.88 ) and 27.65 ( sd 11.36 ) db , respectively . no significant difference was observed between the 2 groups ( student s t - test , p=0.131 ) ( table 6 ) . for caloric testing , 19 of 42 ( 45.2% ) ears showed abnormal results in d group with 11 unilateral cp ( 1 presented with left - sided bppv ) and 4 bilateral cp ( 1 presented with first - degree right - beating spontaneous nystagmus ) . in the nd group , 24 out of 84 ( 28.6% ) ears showed abnormal calorics , with 16 unilateral and 4 bilateral cp . no statistical relationship was found between the calorics results and the appearance of dizziness ( , p=0.06 ) ( table 6 ) . ( , p=0.06 for ac - vemp , p=0.598 for bc - vemp ) ( table 6 ) . to assess the possible relation of the factors of dizziness and caloric results in the measurements of ac 4-pta , bc 4-pta and abg , a 2-way anova was carried out with dizziness having 2 levels [ i.e. , yes ( dizziness present ) and no ( no dizziness ) ] and caloric results having 3 levels ( i.e. , cp at the ipsilateral ear [ the ear in which pta and air - bone gap(abg ) are measured and enter the study ] , cp at the contralateral ear ( so the ear measured has normal calorics ) , and normal calorics ( referring to cases with no canal paresis to either ear ) . for cases with bilateral cp in both ears were counted as ipsilateral cp . concerning ac and bc 4-pta , the anova did not show a significant main effect either for dizziness or for calorics , meaning that there were no significant differences of the means of the above measurements , neither between the dizzy and non - dizzy group , nor between the ears with cp and the ears with normal calorics . however , a statistically significant interaction was found between the 2 factors ( dizziness and calorics ) in relation to ac and bc - pta thresholds . in the dizziness group ( d ) , ears with cp showed much higher means of ac and bc 4-pta , whereas in the non dizziness ( nd ) group the contralateral ear ( with normal calorics ) showed higher ac and bc 4-pta means ( figures 1 and 2 ) . concerning the abg , no significances were found ( p=0.364 for the interactions , p=0.971 , p=0.522 for the main effects of dizziness and calorics , respectively ) . nevertheless , few published studies have attempted to investigate inner ear function and impairment of vemps in otosclerosis . our results , by using ac and bc tone - burst vemps in combination with audiologic findings , are in agreement with the fact that ac - vemp is more vulnerable to conductive hearing loss , whereas bc vemp is not . the magnitude of mean abg , ac 4-pta , and bc 4-pta is likely responsible for the pre - operative difference in elicitation of ac - vemps , confirmed by the significantly lower pre - operative abg in patients with recordable ac - vemps in comparison to those with absent ac - vemps ( 22.23 vs. 27.27 db hl , table 1 ) , lower mean of ac 4-pta ( 47.67 vs. 57.28 , table 1 ) and bc 4-pta ( 25.64 vs. 30.01 , table 1 ) . the magnitude of mean abg , ac , and bc 4-pta appears to have no effect on the pre - operative difference in elicitability of bc - vemps . complementary to this is the finding that the latencies p1 and n1 of bc - vemps in otosclerotic ears are statistically longer than those of normal ears ( table 3 ) , whereas the latencies of ac - vemp are not . this can be explained by the persistence of bc - vemp appearance in ears with advanced otosclerosis , where ac - vemp has already vanished . the low presence rate of bc - vemps pre - operatively ( 44.03% ) could be caused by inner ear damage due to an ototoxic effect of substances produced by otosclerotic foci of the otic capsule on saccular receptors . the fact that the mean of abg differs considerably between ears with present and absent ac - vemp , whereas in ears with bc - vemp it does not ( table 1 ) , reveals a different behavior of the 2 types of vemps in relation to abg values . the different trends concerning the abg means when considering each vemp measurement separately , can explain the lack of statistical significance in the means of abg among the 4 groups formed by the combination of ac and bc - vemp . the same is true for bc 4-pta and ac 4-pta , with the exception that the latter shows a statistically significant difference between the group of ac / bc - vemp both present and ac / bcvemp both absent ( table 4 ) . the small group of 5 ears with present ac - vemp and absent bc - vemp could be explained by the fact that the bone conduction stimulus in the present study was relatively low ( 65 db hl ) , so these could be patients with otolithic organ disease needing a stronger stimulus to produce bc - vemp , whereas the much stronger air - conducted stimulus could do it . in fact , these are ears with relatively low abg means ( 18 db , table 4 ) , so the transition of air - conducted stimuli was not severely hindered . a possible relationship of vertigo and otosclerosis is an understandable assumption , because otosclerosis is a disease affecting structures proximal to the vestibule , but until now , sufficient and concrete evidence supporting this has been lacking . assessment of the functional state of the labyrinth as a total by using a combination of vemp with calorics provided us with interesting observations . firstly , when vertigo / dizziness as a symptom was compared with caloric responses ( not taking otosclerosis into account ) , results were borderline insignificant ( p=0.06 , table 6 ) . any vertiginous patient can have a normally functioning labyrinth with symptoms due to incomplete central compensation or migrainous vertigo . no relation could be found between vemp results and calorics , neither for air- nor for bone - conducted vemps ( table 5 ) . this is an interesting finding , supporting the notion that caloric deficiencies are rather irrelevant to vemp deficiencies in patients with otosclerosis , so it encourages the assumption that vemp deficiencies are due to otosclerosis and are not caused by a history of vestibular neuritis . ac and bc - vemp also did not show any relationship with dizziness either ( table 6 ) , although the percentage of ears with present ac - vemp in the non - dizziness population ( 32.1% ) was at the borders of statistical significance compared with the percentage of ears with present ac - vemp in the dizziness population ( 16.7% ) . there are authors who assume that the balance problems associated with otosclerosis are caused by saccular dysfunction originating from endolymphatic or saccular hydrops [ 3639 ] or direct invasion of the otosclerotic focus to the saccular macula or saccular afferents . a 2012 study by saka et al . found that balance problems in otosclerosis were associated with abnormal results for bc - vemp . conversely , the present analysis of 74 patients with otosclerosis , relying on the assessment of a higher clinical sample than other prospective studies , could not support this theory based on vemp only . a possible involvement of otolithic organs in vertiginous symptoms ( otolithic vertigo ) needs further investigation ( subjective visual vertical and horizontal ) as well as a more detailed questionnaire dealing with the specifics of otolithic vertigo symptoms . anova assessed the relation of vertigo and caloric results , each separately , as well as a combination , with abg and pta measurements ( i.e. , the severity of otosclerosis ) . the results showed no main effect of these 2 factors on the severity of otosclerosis ( abg ) , meaning that the degree of hearing loss was not relevant to either dizziness or caloric results . this is understandable , considering that the semicircular canals are relatively spared from the disease . bppv could be a clinical entity connecting ( not immediately ) saccular involvement with a semicircular canal , but just 1 patient is not enough to support any possible attribution of bppv to otosclerosis . however , anova showed a significant interaction of vertigo and calorics in relation to pta , both air - conducted ( ac 4-pta ) and bone - conducted ( bc 4-pta ) . this means that canal paresis ( cp ) influences pta means differently in the 2 groups of patients . in the group of vertiginous patients ( d ) , ears with canal paresis showed higher pta thresholds ; whereas in the non - vertiginous group ( nd ) , the ears with canal paresis showed lower pta thresholds than ears with normal calorics ( figures 1 and 2 ) . one possible explanation is that although otosclerosis is not a cause of canal paresis or vertigo , if vestibular neuritis ( or hydrops or any other vestibular cause of canal paresis ) affects an ear with advanced otosclerosis , ( i.e. , high threshold of ac and/or bc - pta ) , central compensation is delayed and vertiginous symptoms persist ; whereas in cases of less advanced otosclerosis , compensation is expected to be faster . the principal assumption of the present study is that the mere presence of vemp does not exclude otosclerosis . despite the conductive hearing loss , vemp can still be elicited in a number of preoperative otosclerotic ears . in our study , the response rate of the ac - vemps was only 29.36% , but the response rate of bc - vemps was 44.03% . therefore , a possible suggestion is to proceed to vemp threshold measurements in case of doubt . specifically , positive ac - vemp with pathologically decreased thresholds ( less than 70 db to tone - burst testing at 500 hz , while thresholds for evoking vemp using air - conducted tone - burst in our study were ranging between 95 and 102 db ) excludes middle ear pathology such as otosclerosis and provides a reference for further imaging examination to exclude the alternative diagnosis of scds ( superior canal dehiscence syndrome ) and lvas ( large vestibular aqueduct syndrome ) . therefore , when it is not possible to make an accurate diagnosis based solely on the audiogram and immittance testing results , vemp testing can be added in the diagnostic work - up of otosclerosis to provide additional confirmation . thus , vemp testing outcomes may provide the basis not only for better preoperative counselling , but also for safety issues regarding stapedectomy candidates . the presence of vemp does not exclude otosclerosis . despite the conductive hearing loss , vemp can be elicited in a small number of ears with otosclerosis . the ac - vemp is more vulnerable to conductive hearing loss , whereas bc - vemp is not ; however , the low presence rate of bc - vemps could be caused by inner ear damage . the evaluation of vemp thresholds , by using air - conducted tone - burst , can be added in the diagnostic work - up of suspected otosclerosis in case of doubt , providing additional confirmation , narrowing down the differential diagnosis in patients with pseudo - conductive components , and reducing medical cost by preventing unnecessary radiation exposure and unsuitable middle ear surgery . vertiginous symptoms should not be attributed to otosclerosis itself , but to causes affecting the semicircular canals . perhaps further investigations into otolithic disturbances ( such as subjective visual vertical and horizontal ) and a questionnaire more oriented to otolithic vertigo symptoms could reveal possible clinical impacts of utricular and saccular involvement in the disease . osteoma is a benign osteogenic lesion , which was first described as a specific entity by jaffe in 1935 . in the course of their slow but steady increase in size , osteomas of the maxillofacial bones remain asymptomatic until they attain sufficient sizes as to cause disfigurement and/or direct interference with the normal function of their anatomic location . osteomas are bone lesions with different onset and slow growth that may be divided into : ( 1 ) cranial and mandibular exophytic osteomas ( or eburnean exostoses ) ; ( 2 ) paranasal sinuses , facial bones , and orbit osteomas ( orbital cavity osteoma ) ; ( 3 ) exostoses or bone islands ; and ( 4 ) long bone superficial osteomas ( juxtacortical ) . the etiology seems to be unknown ; however , it may be developmental , infectious , or of traumatic origin . solitary osteomas of the facial skeleton are quite rare , although multiple osteomas of the jaws have been reported as part of gardner 's syndrome . little information is available about the gender predilection ; however , some authors suggest that there is a higher incidence of osteomas in male , especially in between second and fourth decade of life . a 45-year - old male patient was referred to the department of periodontics , krishnadevaraya college of dental sciences and hospital , bangalore , for the evaluation and treatment of a palatal bony swelling [ figures 1 and 2 ] . patient complained of a slow growing , painless mass in the left palatal maxillary area , which was present since six months . he also gave a history of foreign body ( fish bone ) lodgement in 25 and 26 region seven months ago ; however , no treatment was taken for the same . he was systemically healthy with no relevant medical history , and was asymptomatic except for the discomfort in that region and difficulty with mastication . on oral examination , a firm , lobulated , painless , well - circumscribed , exophytic , mass 1.51 cm in diameter was observed on the maxillary posterior left palatal area in relation to 25 and 26 regions . periodontal probing revealed a 9 millimeter deep periodontal pocket associated with the mid palatal area of 26 which prompted us to intervene and surgically excise the bony mass . clinically , the tooth was firm and not associated with any mobility [ figure 3 ] . bony lesion on palate nine millimeter pocket depth a region of well defined opacity was seen in the palatal regions of 25 and 26 [ figure 4 ] , when seen on a maxillary occlusal radiograph . the radio - opacity showed a well defined medial convex margin ; laterally , the margins were indistinct with rest of the structures . region of well defined radio - opacity in the region of 25 and 26 under local anesthesia , buccal and palatal flaps were raised , giving crevicular incisions in 24 , 25 , and 26 regions [ figures 5 and 6 ] . a second vertical incision was placed on the distal of the first premolar and a full thickness flap was raised to allow for adequate exposure of the surgical site . the bony mass was excised with the help of a kirkland chisel which has one end that is a back - action chisel and the other end is a straight chisel [ figures 7 and 8 ] . care was taken to excise the lesion keeping a periphery of healthy tissue around the lesion . the straight end of the kirkland chisel was used to refine the bone margin of the surgical site and to round off any sharp edges . thorough scaling and root planing coupled with removal of the granulation tissue followed by irrigation of the surgical site with saline were performed . margins of the flap were trimmed to achieve good approximation and the flap was sutured with black silk sutures [ figure 9 ] . the specimen was put in a bottle containing 10% formalin solution and sent for histopathological examination . the healing was uneventful , and the sutures were removed after 10 days [ figure 10 ] . the case was followed - up for a period of one year , and there was no sign of recurrence [ figures 11 and 12 ] . sulcular incision given flap raised using vertical incision excision of bony lesion with kirkland chisel section showed parakeratinized stratified squamous epithelium ; sub epithelially a rim of normal connective tissue was seen around the rete ridges . deeper connective tissue showed plump mesenchymal cells with prominent nuclei leading to formation of bony trabeculae . the lesional tissue showed numerous delicately arranged trabeculae with the osteocytes , but was devoid of osteoblastic rimming . the inter - trabecular tissue showed marrow spaces pre - dominantly made up of adipose tissue , presence of few arterioles and capillaries [ figures 13 and 14 ] . histopathologically , the lesion was diagnosed as peripheral ( mature ) osteoma , which is also referred to as ivory osteoma . ( b ) adipose cells ( a ) parakeratinized stratified squamous epithelium ( b ) connective tissue a region of well defined opacity was seen in the palatal regions of 25 and 26 [ figure 4 ] , when seen on a maxillary occlusal radiograph . the radio - opacity showed a well defined medial convex margin ; laterally , the margins were indistinct with rest of the structures . under local anesthesia , buccal and palatal flaps were raised , giving crevicular incisions in 24 , 25 , and 26 regions [ figures 5 and 6 ] . a second vertical incision was placed on the distal of the first premolar and a full thickness flap was raised to allow for adequate exposure of the surgical site . the bony mass was excised with the help of a kirkland chisel which has one end that is a back - action chisel and the other end is a straight chisel [ figures 7 and 8 ] . care was taken to excise the lesion keeping a periphery of healthy tissue around the lesion . the straight end of the kirkland chisel was used to refine the bone margin of the surgical site and to round off any sharp edges . thorough scaling and root planing coupled with removal of the granulation tissue followed by irrigation of the surgical site with saline were performed . margins of the flap were trimmed to achieve good approximation and the flap was sutured with black silk sutures [ figure 9 ] . the specimen was put in a bottle containing 10% formalin solution and sent for histopathological examination . the healing was uneventful , and the sutures were removed after 10 days [ figure 10 ] . the case was followed - up for a period of one year , and there was no sign of recurrence [ figures 11 and 12 ] . section showed parakeratinized stratified squamous epithelium ; sub epithelially a rim of normal connective tissue was seen around the rete ridges . deeper connective tissue showed plump mesenchymal cells with prominent nuclei leading to formation of bony trabeculae . the lesional tissue showed numerous delicately arranged trabeculae with the osteocytes , but was devoid of osteoblastic rimming . the inter - trabecular tissue showed marrow spaces pre - dominantly made up of adipose tissue , presence of few arterioles and capillaries [ figures 13 and 14 ] . histopathologically , the lesion was diagnosed as peripheral ( mature ) osteoma , which is also referred to as ivory osteoma . ( a ) trabeculae with osteocytes ( b ) adipose cells ( a ) parakeratinized stratified squamous epithelium ( b ) connective tissue in gnathic bones , osteoma can appear in bone 's surface , as mushroom - like masses pedunculated or sessile ( periosteum osteoma ) , or located in medullary space ( endosteum osteoma ) . these lesions are usually asymptomatic , but may proliferate in medullary bone ( endosteal ) or on the bone surface as a polypoid or sessile mass ( periosteal ) . reactive bone growth from infection , trauma , or vascular malformation may produce lesions simulating an osteoma . these lesions may arise at any age but with a distinct predilection for elderly adults and they are twice more common in male than in female . as a pathologic entity , osteoma is difficult to differentiate radiographically from other lesions with pronounced ossifications . exostosis , which is the term generally applied for well demarcated and extremely opaque bony lesion , osteosclerosis and similar alterations of the bone are always discussed alongside osteomas . the other differential diagnosis include enostosis , cementoma , complex odontome , fibrous dysplasia , osteochondroma , paget 's disease , peripheral ossifying fibroma , a reactive focal lesion ; periosteal osteoblastoma , osteoid osteoma , and paraosteal osteosarcoma that present as painful masses of rapid growth.[79 ] though the exact etiology is not known , it may be suggested , in this case that trauma from foreign body lodgment as afore mentioned might have been the trigger for this proliferative response . osteomas are slow growing benign osteogenic lesions arising from the proliferation of either cancellous or compact bone . they are usually asymptomatic and have a low recurrence rate . simple excision of the lesion in its entirety is the treatment of choice in symptomatic lesions . the association of osteoma with gardner 's syndrome must always be kept in mind before deciding the treatment plan .

backgroundthe aim of this prospective clinical study was to evaluate the clinical importance of vestibular - evoked myogenic potentials ( vemps ) in the assessment and differential diagnosis of otosclerosis and otologic diseases characterized by pseudo - conductive components . we also investigated the clinical appearance of balance disorders in patients with otosclerosis by correlating vemp results with the findings of caloric testing and pure tone audiometry(pta).material / methodsair - conducted(ac ) 4-pta , bone - conducted(bc ) 4-pta , air - bone gap(abg ) , ac , bc tone burst evoked vemp , and calorics were measured preoperatively in 126 otosclerotic ears.resultsthe response rate of the ac - vemps and bc - vemps was 29.36% and 44.03% , respectively . statistical differences were found between the means of abg , ac 4-pta , and bc 4-pta in the otosclerotic ears in relation to ac - vemp elicitability . about one - third of patients presented with disequilibrium . a statistically significant interaction was found between calorics and dizziness in relation to pta thresholds . no relationship was found between calorics and dizziness with vemps responses.conclusionsac and bc vemps can be elicited in ears with otosclerosis . ac - vemp is more vulnerable to conductive hearing loss . evaluation of ac - vemp thresholds can be added in the diagnostic work - up of otosclerosis in case of doubt , enhancing differential diagnosis in patients with air - bone gaps . otosclerosis is not a cause of canal paresis or vertigo . osteomas are benign slow growing , osteogenic lesions which may arise from proliferation of either cancellous or compact bone . they are usually sessile tumours composed of dense sclerotic , well formed bone projecting out from the cortical surface , most often of the skull and facial bones . this paper reports a case of a peripheral osteoma in the hard palate of a 45-year - old man , which was treated by periodontal flap surgery with surgical excision of the bony lesion . peripheral osteomas of jaw bone are uncommon and usually associated with gardner 's syndrome . histological examination confirmed the clinical impression of a peripheral osteoma . patient was reviewed after one year and was asymptomatic with no recurrence of the lesion .

fig fruit represents an important constituent of the diet , because of their nutritional and medicinal values . this type of diet is considered one of the healthiest and is associated with longevity . figs are an excellent source of minerals , vitamins , polyphenols , and dietary fiber ; they are fat and cholesterol - free and have a high antioxidant activity . figs have recently attracted a great deal of attention and are widespread throughout the world . the world produces over one million tons of figs yearly , of which 82% are produced in mediterranean countries . in tunisia , the production is about 29 000 tons ; it represents 3% of total world production . the fig is an economically important fruit species which is cultivated extensively in southeast regions . fig nursery plants production is mainly based on cuttings taken from the mother tree and placed in a rooting , which will eventually produce roots and shoots [ 68 ] . the traditional method for growing fig consisted in taking cuttings from one- or two - year - old shoots and directly planting them in soil to obtain new trees . however , the investigation on ways to propagate fig tree material and produce vigorous and healthy fig plants under field conditions becomes necessary in order to develop fig sector . the objective of this study was to characterize and compare fig nursery plants of some local fig cultivars by using hardwood cuttings under field conditions . in this way , five cultivars and six cuttings sizes were compared in order to optimize the propagation method of fig in tunisia . in this research , hardwood cuttings of 4 uniferous ( one production per year ) fig cultivars ( bayoudhi , jemaoui , ragoubi , and zidi ) , one biferous ( two productions per year ) ( bither ) , and one caprifig ( male fig ) ( bouharrag ) were used as plant material . fig tree is a species having high level of adaptation to different soil types and dry conditions . these cultivars are frequently cultivated for producing fresh and sun drying fig in arid region of tunisia ( 100200 mm annual precipitation ) . these ones are widely grown in southern tunisia and were described in a previous work [ 10 , 11 ] . hardwood cuttings were taken at the beginning of february 2009 and february 2010 , from adult fig trees of the germplasm collection of arid land institute ( ira)medenine , located in el gordhab tataouine ( tunisia ) . it consists in comparing the ability of cutting of 5 cultivars ( bayoudhi , jemaoui , ragoubi , zidi , and bouharrag ) . ten cuttings per replicate and per cultivar were used , with a total of 150 cuttings . it consists in comparing the ability of cutting of 5 cultivars ( bayoudhi , jemaoui , ragoubi , zidi , and bouharrag ) . ten cuttings per replicate and per cultivar were used , with a total of 150 cuttings . it was involved on the cultivar bither with 6 cuttings types as described below : type 1 : cuttings of one - year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 2 : cuttings of one - year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 3 : cuttings of one - year - old shoot , length ( 60 cm ) , and diameter ( < 1.5 cm ) ; type 4 : cuttings of 2-year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 5 : cuttings of 2-year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 6 : cuttings of 2-year - old shoots , length ( 60 cm ) , and diameter ( > 2 cm ) . it was involved on the cultivar bither with 6 cuttings types as described below : type 1 : cuttings of one - year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 2 : cuttings of one - year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 3 : cuttings of one - year - old shoot , length ( 60 cm ) , and diameter ( < 1.5 cm ) ; type 4 : cuttings of 2-year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 5 : cuttings of 2-year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 6 : cuttings of 2-year - old shoots , length ( 60 cm ) , and diameter ( > 2 cm ) . type 1 : cuttings of one - year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 2 : cuttings of one - year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 3 : cuttings of one - year - old shoot , length ( 60 cm ) , and diameter ( < 1.5 cm ) ; type 4 : cuttings of 2-year - old shoot , length ( 30 cm ) , and diameter ( < 1.5 cm ) ; type 5 : cuttings of 2-year - old shoot , length ( 40 cm ) , and diameter ( < 1.5 cm ) ; type 6 : cuttings of 2-year - old shoots , length ( 60 cm ) , and diameter ( > 2 cm ) . ten cuttings per replicate and per cutting types were used , with a total of 180 cuttings . during this research , cuttings were planted directly in rooting units ( 2.5 0.65 m ) under field conditions . cuttings were planted in an inclined position ( angle of 45 ) on sandy soils , where 3/4 of cutting is sinking into the soil at the professional agricultural training center ( cfpa ) el gordhab tataouine ( tunisia ) . all plants were irrigated by submersion technique with 40 l / rooting unit/15 days on the early morning during the course of the experiment in the three seasons . maintenance works such as weed control and hoeing were carried out during the whole vegetation period . root emergence percentage , number of emerged buds / nursery plant , length of nursery plant from the soil surface , and diameter of nursery plant were measured on the section 5 cm higher than soil surface and number of leaves / nursery plant . at the drawing out of young plants , cumulative growth ( cutting length + new growth ) of nursery plant and number of roots / nursery plant were determined [ 12 , 14 ] . all parameters were measured at the end of the vegetation period ( almost 8 months after plantation ) . emerged buds , leaves , and roots of each plant were counted manually . data were subjected to analysis of variance ( anova ) , using statbox version 6.40 . in case of significant cultivars and cutting type 's effects , comparison of values was performed by means of the least significant difference ( lsd ) test at a significance level of 0.05 , as the percentage root emergence ( re % ) was calculated by the means of three replications . the descriptive data analysis revealed a high variability among all the morphological characters of young plant fig cultivars studied under the field conditions . the most widely varied characters were root emergence percentage ( 10%90% ) and cumulative growth of nursery plant ( 32112 cm ) . considerable range of variation was also noticed in length of nursery plant ( 4 to 70 cm ) and number of leaves / nursery plant ; it varied from 3 leaves to 63 leaves , whereas very low variation was noted in number of emerged buds / nursery plant , diameter of nursery plant , and number of roots / nursery plant ( table 2 ) . the mean values of all morphological characteristics observed were statically different at a 5% level ( table 3 ) . this indicates a certain variation within cultivars , especially for length , diameter of nursery plants , number of leaves , and cumulative growth of nursery plants . root emergence percentage ( % re ) had the highest values ( 65% ) in jemaoui and rogabi cultivars and the lowest values ( 15% ) in bayoudhi . nursery plants characteristics like length of nursery plant ( lp ) , diameter of nursery plant ( dp ) , cumulative growth of nursery plant ( ag ) , and number of roots / nursery plant ( nr ) exhibit the highest values in cultivars bayoudhi . finally , the number of leaves / nursery plant ( nl ) was significantly high in cultivars bouharrag ( 37 leaves ) and bayoudhi ( 27 leaves ) . the result of this part can provide that the cultivars choice is very important to produce sufficient amounts of vigorous nursery plants . cultivar is very adapted to agroecological conditions , which can be eventually developed by farmers . moreover , the length of nursery plant ( lp ) , diameter of nursery plant ( dp ) , and number of roots ( nr ) are very important quality characters for selection of nursery plants of fig cultivars . results of the descriptive data analysis recorded high morphological variation in the plant material derived from the 6 fig cutting types of the cultivar bither , for all the traits . the most widely varied parameters were root emergence percentage ( 090% ) , length of nursery plant ( 377 cm ) , and number of roots / nursery plant ( 029 roots ) ( table 4 ) . results of 6 cutting types for the cultivar bither analysis showed a significant difference at a level of 5% in all the studied characters ( table 5 ) . the length of nursery plant ( lp ) , diameter of nursery plant ( dp ) , cumulative growth of nursery plant ( cg ) , and number of roots / nursery plant ( nr ) showed high significant difference . results revealed that root emergence percentage ( re % ) had the highest value in cutting type 3 and the lowest value in type 6 . also , type 3 exhibits the highest means of number of roots / nursery plant ( nr ) , length of nursery plant ( lp ) , and number of leaves / nursery plant ( nl ) . the cutting type 5 was significantly high in the cumulative growth trait in nursery plant ( cg ) . finally , the number of roots / nursery plant ( nr ) presents the highest value in cutting type 5 . the findings of the study put forth that cutting characteristics ( length , diameter , and age ) affected the growth and quality of fig plants . moreover , cutting length affected the length ( lp ) and cumulative growth ( cg ) of fig nursery plant . the use of long cuttings ( 4060 cm ) is recommended to produce vigorous fig nursery plants . the results achieved in this study showed that propagation of fig plant material under field conditions can be applied and should not require sophisticated equipment [ 6 , 12 ] . the values of characters measured on nursery fig plants in the present study were compared with those from other countries , namely , morocco [ 12 , 15 ] , turkey [ 14 , 16 ] , iran , thailand , and chile . similar percentage of rooted cuttings on some moroccan varieties propagated by using hardwood stem cutting under field conditions was found . chaari , aqounaq el hmam , ghouddane , ournaki , and kadota presented high values of % root emergence ; 89 , 98 , 94 , 87 , and 88% , respectively . cultivar sarilop from turkey has revealed high similarity in morphological characteristics of nursery plants with bayoudhi and jemaoui cultivars from the present study . in turkey , the fig cultivar calimyrna sarilop is used and propagated extensively by cuttings and calimyrna is the most popular and best cultivar in the world because it dries well . nevertheless , rooting ratio % , length of nursery plant , and diameter of nursery plant reported lower values than those of tunisian cultivars studied . the iranian cultivar sabz represents the large majority of fig trees present in estahban area . obtained values on root emergence percentage , number of emergence buds , length of nursery plant , and number of leaves / nursery plant variables are similar to our results . brown turkey , dauphine , and lisa cultivars from thailand showed variables values of nursery fig plants comparable with those of this work . however , root emergence percentage revealed lower values ( 12.5039.29% ) than that found in the present survey ( 1583.33% ) , whereas three cultivars showed higher values for the number of emerged buds and number of roots / nursery plant . moreover , the high variation was recorded among the % roots emergences and the other morphological traits within the 5 cultivars ( bayoudhi , jemaoui , ragoubi , zidi , and bouharrag ) and between the 6 different types of cuttings ( age , length , and diameters ) of the cultivar bither . these variations were influenced by ability of cultivar for rooting and characteristics of cuttings ( length , diameter , and age ) . furthermore , practices of farming ( soil preparation , irrigation , harrowing , etc . ) and climate conditions were cited to have an effect on the quality of fig plants in the nursery [ 13 , 18 ] . several factors seem to be involved in the improvement of fig hardwood cutting like the use of rooting hormonal treatment and the use of plastic tunnels in fig propagation . the present research showed that the hardwood cuttings of local cultivars of figs can be used to propagate material in field conditions . furthermore , it is possible to obtain vigorous young plants ( length , number of leaves , and cumulative growth ) from cuttings of cultivars bouharrag ( caprifig ) , bayoudhi ( common fig ) , rogabi , and zidi ( smyrna fig ) . bither 's rooting ability of the 6 cutting types studied revealed that the cutting type of one - year old characterized by 60 cm of length and diameter of < 1.5 cm gave the best percentage of rooting emergence . also , the best cumulative growth of plant was obtained with the cutting type ( 2 years old shoots , length ( 40 cm ) , and diameter ( < 1.5 cm ) ) . the positive aspects of this experiment have demonstrated the suitability of the hardwood cuttings method for vegetative propagation , since the production of fig plants in nursery can be realized and seems to be cheap . this work is supposed to be a preliminary positive contribution in the propagation of fig in nursery which can have practical application for fig horticulturists in the southeast of tunisia . unintentional durotomy is a frequent complication of spinal surgical procedures , with a rate as high as 17% ( 1 ) . a small percentage of patients with unintentional durotomy sustain intracranial haemorrhage , likely due to an alteration in intradural hydrostatic dynamics caused by cerebrospinal fluid hypovolaemia . despite the gravity of this complication , to our knowledge , only a few cases have been previously documented . to our knowledge a case of new onset epilepsy has never been reported in the literature . the purpose of this report is to describe a case in which new - onset tonic - clonic seizures occurred following an unintentional durotomy during lumbar discectomy and to discuss the importance of our findings in relation to the possible pathophysiological mechanisms . a 62 year old female presented to a spinal orthopaedic clinic complaining of pain in the anterior aspect of her right thigh for at least a year . she could only walk for 15 minutes , thus this limited the patient s activities of daily living . medical history was unremarkable but for bipolar depression which was treated by electroconvulsive therapy and anti - psychiatric medication . 14 ) showed l2/3 disc prolapse impinging on the right exit canal and causing l2 and l3 compression , thus she was listed for l2/3 lumbar discectomy and decompression . examination of the right l2/l3 dermatomes revealed severe paresthesia and mild grade 4/5 weakness ; reflexes were all normal . preoperative sagittal t1 weighted image showing disc bulge at l2/3 preoperative sagittal t2 weighted image showing disc bulge at l2/3 elective bilateral l2/l3 lumbar discectomy and l2/l3 decompression was performed . preoperative axial t2 weighted image showing disc bulge at l2/3 to the right . during dissection , this was primarily sutured and covered with duragen ( integra lifesciences , plainsboro , new jersey ) . post - operatively the patient was well ; she experienced a few headaches but still had no neurological deficit . the patient was recovering well when suddenly , 48 hours post - operatively , the patient experienced four generalized tonic - clonic seizures , and was deeply post - ictal following the seizures . a diagnosis of tonic - clonic seizures secondary to low intracranial pressure was made . all routine blood investigations as well as blood amisulpride and olanzapine levels carried out on all post - operative days were found to be within normal limits . a post - operative head / brain computed tomographic ( ct ) scan showed no infarct , haemorrhage or mass lesions . the patient was started on oral phenytoin 3mg / kg daily and had no further episodes of seizures . she never reported any problems during the duration of follow - up and was successfully discharged back to the community . dural tears during spinal surgery are not uncommon , with reported incidence rates of 0.3% to 17% ( 1 , 2 ) . problems arising from this complication are rare , but devastating and require astute recognition of the early signs of neurological impairment . here we are attempting to make an association between the csf hypotension caused by an unintentional durotomy at lumbar surgery and the new onset of epilepsy which might have been propagated by the patients pre - operative anti - psychiatric medication . the phenomenon of intracranial haemorrhage remote from a surgical site has been described in the literature , and several authors have attempted to explain the pathophysiology ( 1 , 2 ) . although intracranial haemorrhage did not occur in this case , the same pathophysiology is thought to have contributed to provoke the new onset epilepsy . the so - called sag model postulates that haemorrhage results from altered cerebrospinal fluid hydrodynamics , causing caudal sagging of the cerebellum with stretching of cerebellar vermian veins ( 1 ) . taking this pathophysiology into account this was satisfactorily repaired intra - operatively , however , the csf leak was substantial and the dura may have been weakened and prone to later leak . we hypothesize that this may have resulted in delayed intracranial hypotension , causing primary tonic - clonic seizures and not an intra - cranial bleed . thus , we believe that the hypotensive insult coupled with an already lowered seizure threshold , due to the patient s long term anti - psychiatric medical history , gave rise to these new onset tonic - clonic seizures . we are unaware of any cases of new onset epilepsy post - durotomy . although dural rupture during surgery and csf hypovolemia are thought to be the main contributing factors to the above , we have great reservations about the types of chronic medications the patient was having . one may only postulate about the association between both olanzapine and amisulpride and their epileptogenic side effects . in the british national formulary a case report published by woolley and smith ( 3 ) shows that atypical neuroleptics like olanzapine and amisulpride lower seizure threshold . manufacturer 's trials gave a seizure rate , similar to other antipsychotics , of 0.88% patients ( product data sheet , eli lilly ) . in this article it is stated that the patient in question represents the strongest case to date implicating olanzapine alone in lowering seizure threshold , with objective eeg support . the manufactures data for both drugs issue warnings in their literature stating that both amisulpride and olanzapine can lower seizure threshold and therefore should be used with caution in patients previously diagnosed with epilepsy . however , they do not state that in cases where the seizure threshold is already lowered for example by csf hypotension , new onset epilepsy might be more common ( 4 ) . in conclusion , new onset seizures following dural tears might appear in patients on anti - psychiatric medication . we strongly recommend that patients on potentially epileptogenic medication are either changed to other forms of anti - psychiatric non - epileptogenic medication or are closely monitored post - operatively in the event of this rare complication developing .

this research was carried out in southeast of tunisia in 2009 and 2010 , in order to study the propagation of six ( ficus carica l. ) cultivars by using hardwood cuttings under the field conditions . the effect of the cultivars and the type of buds , shoots age , shoots length , and shoots diameter were recorded . ten cuttings per cultivar and/or cutting types with three replications were planted in rooting unit . percentage of root emergence and six morphological parameters of young fig plants were measured . results showed that the responses of cuttings as fig nursery plants presented a high variability among the five cultivars . the most widely varied characters were % root emergence ( re ) and cumulative growth of young plant ( cg ) . the first one ranged from 10% to 90% , the second varied within 32 and 112 cm . concerning the bither cultivar , 6 cutting types with different age , length , and diameter were evaluated . results showed a great variation in % of root emergence ( 090% ) , length of nursery plant ( 377 cm ) , and number of roots / nursery plant ( 029 roots ) . the present research showed that the hardwood cutting of local fig cultivars can be propagated under field conditions in southeast of tunisia . we would like to present a rare case report describing a case in which new - onset tonic - clonic seizures occurred following an unintentional durotomy during lumbar discectomy and decompression . unintentional durotomy is a frequent complication of spinal surgical procedures , with a rate as high as 17% . to our knowledge a case of new onset epilepsy has never been reported in the literature . although dural tears during surgery and csf hypovolaemia are thought to be the main contributing factors , one postulates on the effects of anti - psychiatric medication with epileptogenic properties . amisulpride and olanzapine can lower seizure threshold and should be used with caution in patients previously diagnosed with epilepsy . however manufacturers do not state that in cases where the seizure threshold is already lowered by csf hypotension , new onset epilepsy might be commoner . finally , strong caution and aggressive post - operative monitoring is advised for patients with csf hypotension in combination with possible epileptogenic medication .

it seems that complications of vitamin d deficiency have been completely removed by having enriched food by vitamin d , but rachitism is the top of iceberg of vitamin d deficiency . vitamin d deficiency can also causes osteopenia , osteoporosis , and muscle weakness and increases risk of hip fracture in adults . the most important effect of vitamin d is on parathyroid and bone and also plays a key role on the calcium absorption from the gut . the discovery of vitamin d receptor in most tissues and organs has developed a new perception from effects of vitamin d. for instance , vitamin d can decrease risk of many chronic diseases , cancers , autoimmune disease , infectious , and cardiovascular disease . it has been estimated that one billion people are vitamin d deficient in the world . this deficiency is common in all different cities with the same level of the sea or cities with lower geographic latitude . 20 ng / ml is the level of vitamin d deficiency and 20 < 25 ( oh ) d < 30 ng / ml is recognized as a vitamin d insufficiency . there are different ways to detect cut - off points for vitamin d deficiency . for instance , based on inverse relation between 25 ( oh ) d and pth , a direct relation between 25 ( oh ) d and 1 , 25 ( oh ) d and effect of vitamin d on fracture risk . the level of 25 ( oh ) d that would be enough for pth suppression , probably , is a suitable criterion for bone health and cut - off point for vitamin d deficiency . the aim of this study was detection of prevalence of vitamin d deficiency in secondary students in arak , a centrally located city in iran , and also detection of cut - off points for vitamin d deficiency . in a cross - sectional study in november 2010 , 420 students including 220 girls and 200 boys from secondary students from 10 to 16 years old were selected by a multistage sampling . first , all of the secondary schools were divided into two areas and then 21 schools including 11 girls schools and 10 boys , schools were selected by a cluster sampling . finally , the samples were picked up by a systematic random procedure based on the student 's initials . all the students and their parents were informed about the study and signed off the consent form . inclusion criteria were lack of hepatic , renal , bone disease and cancer , no history of intake of anticonvulsant drugs , calcium , vitamin d , and antineoplastic drugs . laboratory tests including hepatic , renal , calcium , phosphoros , alp ( alkaline phosphatase ) , albumin , pth , 25 ( oh ) d. 25 ( oh ) d levels by ria and pth by irma were measured ( made by bio source europe sa , belgium ) and its intensity categorized into three levels . mild deficiency : 15 25 ( oh ) d < 20 ng / ml.moderate deficiency : 8 25 ( oh ) < 15 ng / ml.severe deficiency : 25 ( oh ) < 8 ng / ml . mild deficiency : 15 25 ( oh ) d < 20 ng / ml . moderate deficiency : 8 25 ( oh ) < 15 ng / ml . severe deficiency : 25 ( oh ) < 8 ng / ml . the relation between vitamin d and pth was assessed by the scatter chart . the level of vitamin d that pth was increasing , defined as cut - off points for vitamin d deficiency . the prevalence of vitamin d deficiency in different intensities was assessed by spss 15 and variables compared between girls and boys , and also between deficient and sufficient vitamin d groups by student 's test or mann -- whitney u based on normal distribution based on k -- s ( kolmogrov -- smirnov ) test . the pearson test was used to identify the relation between 25 ( oh ) d and age , weight , and bmi . a total of 420 students including 46.9% boys and 53.1% girls participated in this study . mean age for boys and girls was 12.5 1 and 12.7 1 years old respectively p = 0.1 [ table 1 ] . mean characteristic of students ( mean sd ) mean age of vitamin d - deficient and nondeficient groups based on cut - off 25 ( oh ) d < 20 ng / ml were 12.6 1 and 12.6 2 years old respectively ( p = 0.5 ) . but mean bmi in two groups were 20 3.5 and 19 2.1 respectively ( p = 0.02 ) . there was no meaningful relation between 25 ( oh ) d and age ( p = 0.5 ) but there was an inverse relation between 25 ( oh ) d level and bmi ( r = -0.17 ) ( p = 0.01 ) , weight ( r = -0.14 ) ( p = 0.05 ) , pth ( r = -0.262 ) ( p = 0.001 ) . there was also a meaningful relation between vitamin d deficiency and sex ( p = 0.001 ) . based on the logistic regression analysis , the girls have 54.4 times more chance than boys for vitamin d deficiency ( or 54.4 ) ( p < 0.001 ) . totally 84% of the students had vitamin d deficiency [ table 2 ] . three cut - off points 33 , 21 , and 13 ng / ml were defined for mild , moderate , and severe vitamin d deficiency [ figure 1 ] using the scatter chart . the prevalence of vitamin d deficiency based on different intensities was evaluated in both genders [ table 3 ] . prevalence of vitamin d deficiency in students ( number/% ) mean of pth of different cut - off points for vitamin d deficiency prevalence of vitamin d deficiency based on local cut - off points ( % ) our findings in arak , a centrally located city of iran , showed a high prevalence of vitamin d deficiency of 84% among the students . the results showed that females were more likely to be vitamin d - deficient than males . the present study suggests three local cut - off points for vitamin d deficiency based on 25 ( oh ) d < 13 , 21 , and 33 ng / ml . vitamin d deficiency is a common medical problem throughout the world . it has been predicted that one billion people throughout the world are vitamin d deficient . prevalence of vitamin d deficiency in the countries where food is enriched by vitamin d ( scandinavian countries ) is 1.614.8% . in other european countries , among middle - aged and elderly people , 14 - -59.6 in tunessia 47.6% and finally among teenagers in boston was 24.1% . a total of 30 - -50% of people in india , lebanon , and turkey and also 45.2% of females in china were vitamin d deficient . several studies in different parts of iran and among different age groups have shown a high prevalence of vitamin d deficiency.[1114 ] prevalence of vitamin d deficiency in tehran ( the capital city of iran ) was 60% in males and 91% in females . the prevalence of vitamin d deficiency in isfahan , another central city of iran , was 46.2% among students including 72.1% girls and 18.3% boys and is similar to our findings in tabriz , another city in the north west of iran , which was 64.2% among females ( mild 33.7% , moderate 15.5% , severe 15.1% ) that in comparison with the present study is lower . another study in different cities of iran , with different climate , showed that 27.2% of females and 37.25% of males had a mild vitamin d deficiency , and also 47.85% of females and 34.75% of males had moderate to severe vitamin d deficiency that confirms the present study . different etiologies , such as insufficient sun exposure , clothing habits , and insufficient intake of vitamin d , may explain high prevalence of vitamin d deficiency . it has been shown that 2-years intake of calcium and vitamin d , increased serum 25 ( oh ) d level up to 28% . serum 25 ( oh ) d levels were lowest in blacks and highest in white people . although some studies found that sun expose and clothing habits were not different between sufficient and deficient groups . decreased calcium intake lead to increased serum pth level and increased catabolism of 25 ( oh ) d. awumey et al . showed higher activity of 24-hydroxylase in fibroblasts that lead to increased catabolism of 25 ( oh ) d of indian americans . the darker skin of the asian immigrants to european countries is another risk for vitamin d deficiency . found that 61% of the turkish group ( in turkey ) and 55% of the turkish immigrant group in germany had bone and muscle pain , while it was 15% within the german group with higher serum 25 ( oh ) d levels . our findings showed that the prevalence of vitamin d deficiency is much more in girls than boys ( girls 99.1% , 66.5% boys ) ( p < 0.001 ) . another study in iran , isfahan , also found that the prevalence of vitamin d deficiency was four times more in girls than boys and severe vitamin d deficiency in girls and boys were 14.5% and 0.6% respectively . another study in iran also found that moderate - to - severe vitamin d deficiency was much more in females than males . differences according to gender and age group maybe because of biological differences but might also due to behavioral differences . there might be gender - specific clothing differences ( necessarily not based on a culture religious ) . there also might be that females spend less time outside , resulting in less exposure to the sun . another study in the united states among female soldiers found that vitamin d levels decrease during military training in the summer due to the type of clothing worn during training , coupled with inadequate vitamin d intake . our study found that vitamin d deficiency was not associated with the age , while most studies found higher prevalence of vitamin d deficiency among elderly , probably due to lower capacity of the skin to produce vitamin d after sun exposure or less vitamin d intake in elderly , although a few studies reported higher prevalence of vitamin d deficiency in young people . our study shows a significant inverse association between bmi ( body mass index ) and weight in one hand with vitamin d deficiency in another hand , same as the tabriz study and against the isfahan study . considering the role of fat tissue as a depot of vitamin d , obese have increased storage capacity of 25 ( oh ) d , that leads to lower serum vitamin d levels . we determined three cut - off points for vitamin d deficiency based on 25 ( oh ) d < 33 , 21 , and 13 ng / ml for mild , moderate , and severe vitamin d deficiency , using scatter chart , versus 12.5 , 25 , and 40 ng / ml in another study in iran and 32 ng / ml in isfahan . high prevalence of vitamin d deficiency probably could be the result of above - mentioned combination . we recommend that some efforts could be made to encourage people to intake vitamin d and calcium supplements and also have more exposure to the sunlight . prior to the availability of potent antiretroviral ( arv ) therapy , human immunodeficiency virus type 1 ( hiv-1 ) infection was notable for the inevitable progression to acquired immunodeficiency syndrome ( aids)-defining events and death . with the advent of mono- and dual - nucleoside reverse transcriptase inhibitor ( nrti ) therapy , there was recognition of diverse drug - related adverse events . the introduction of combination arv therapy with dual nrtis plus protease inhibitors ( pis ) was associated with a dramatic decline in aids and mortality , along with an increased appreciation for the development of diverse metabolic complications , such as insulin resistance and dyslipidemia , as well as more recent reports actually showing evidence of premature cardiovascular disease . in addition , co - morbid conditions such as renal and hepatic disease increasingly influenced the quality of the lives of hiv-1-infected individuals . over the ensuing years research has attempted to define the potential role that select arv agents and hiv-1 infection itself have on hepatic , renal , and cardiovascular disease . numerous factors are known to be associated with increased risk of atherosclerotic disease , such as diabetes mellitus , hypertension , smoking , family history and dyslipidemia , all of which occur with variable frequency in those with hiv-1 infection . as combination arv therapy allowed hiv-1-infected individuals to live longer , moreover , concerns are enhanced by the association between select arv agents and insulin resistance , dyslipidemia and fat maldistribution , along with case reports of premature cardiovascular disease . while there is little evidence clearly demonstrating an association between any specific drug and visceral adiposity , lipoatrophy does appear to be a hiv-1-specific condition primarily linked to the use of thymidine analogues such as zidovudine and stavudine . similarly , it is now clear that lipid abnormalities have been seen with select nrtis , non - nrtis , and pis . in fact , there are specific guidelines for the management of dyslipidemia in hiv-1-infected individuals . while several studies have reported a relationship between arv use and cardiovascular events , the d : a : d ( data collection on adverse events of anti - hiv drugs ) cohort is the largest longitudinal study specifically designed to address this question . this study has provided the best evidence that there is an independent relationship between duration of combination arv therapy and cardiovascular events , with most of the effect initially being linked to the use of pis . more recent analyses of this cohort have addressed the relationship between nrti use and cardiovascular disease . here they showed that the recent use of abacavir and , to a lesser extent , didanosine was associated with increased risk of cardiac events when compared to regimens that did not use these drugs . notably the absolute risk was small for all groups and was most prominent amongst those with other cardiovascular risk factors . a follow - up analysis presented at the 16 conference on retroviruses and opportunistic infections showed that there was also an independent association between cardiovascular events and the use of lopinavir / ritonavir and indinavir , while no such relationship was seen with tenofovir disoproxil fumarate ( tenofovir df ) or the non - nrtis efavirenz and nevirapine . it is important to note that while cohorts can include large numbers of longitudinally followed patients , they are not randomized controlled trials and therefore have numerous limitations . that being said , a similar association between abacavir use and cardiovascular events was recently reported from the smart ( strategies for management of anti - retroviral therapy ) study and a case control study of patients in the anrs ( national agency for aids research ) french hospital cohort . nevertheless , questions remain since there has been no definitive biological explanation given for these findings and other studies have not demonstrated such associations . intriguing data have recently emerged to suggest a relationship between ongoing viremia and cardiovascular disease . data from the smart study , designed to assess whether treatment interruption in those with higher levels of cd4 t cells could minimize some of the toxicity associated with arv therapy , actually showed overall increased risk of several non - aids events , including cardiovascular , renal and hepatic disease , amongst those that interrupted treatment . further analyses from the smart study have demonstrated that markers of inflammation , such as interleukin-6 ( il-6 ) , and coagulation , such as d - dimers , are increased in those off arv therapy , all of these markers having been shown in other populations to be associated with increased risk of cardiovascular events . stored samples from the smart study have further been used to link these changes in markers of inflammation with abacavir use . in a cross - sectional analysis of patients either on or off abacavir at baseline , the investigators showed increased levels of high sensitivity c - reactive protein and il-6 in those on abacavir compared to those not taking this drug . however , another study showed different results , demonstrating that related markers declined to a similar extent in both arms in a randomized controlled trial comparing tenofovir df / emtricitabine to abacavir / lamivudine . these differences may reflect the difficulty in observing a potentially modest relationship between select arv agents and markers of inflammation in the face of the overwhelming influence that suppressing plasma hiv-1 rna has on these same measures . while results are inconclusive , other potential markers associated with increased cardiovascular disease have also been evaluated as explanations for the possible relationship between select arv agents and cardiovascular events , such as endothelial function and platelet aggregation . while there remains considerable uncertainty as to how these various data sets should be interpreted , these observations have led to new research exploring the relationship between hiv-1 replication and potential pathogenic mechanisms for select end - organ events . renal disease is common amongst those with hiv-1 infection and is often multifactorial ; possible factors include hiv-1-associated nephropathy and co - morbid conditions such as diabetes mellitus and hypertension , as well as co - infection with hepatitis b and c . certain arv drugs have also been associated with nephrotoxicity , such as indinavir , a drug rarely used in the current era that frequently caused nephrolithiasis and occasionally interstitial nephritis . in addition , tenofovir df has been linked to the development of proximal renal tubular dysfunction . it is clearly recommended that routine monitoring of renal function should occur in all hiv-1-infected patients , with particular attention given to those with co - morbid conditions or taking nephrotoxic drugs . one recent study attempted to define the relationship between renal function , tenofovir df use , and the degree of plasma hiv-1 rna suppression . this was a relatively small cohort study showing that those with complete viral suppression on a tenofovir df - containing regimen actually experienced an increase in glomerular filtration rate . in contrast , there was a small but significant decline in renal function amongst those on tenofovir df that did not achieve full virologic suppression . the authors hypothesize that ongoing viremia , and perhaps the associated increase in inflammation , could be contributing to these adverse outcomes . other studies have shown a similar relationship between hiv-1 replication and progression of renal disease . the overwhelming burden of hepatic disease in hiv-1-infected individuals is related to co - infection with hepatitis b and c . hiv-1 and hepatitis co - infection treatment guidelines provide detailed information about relevant interactions between these chronic viral infections and how co - infection influences the management of each pathogen . there is also an increased risk of hepatic steatosis that may be associated with hyperlipidemia , insulin resistance and select arv agents . several arvs have also been shown to result in hepatotoxicity , the strongest links being with high - dose ritonavir , rarely used in the current treatment era , and tipranavir and nevirapine , the latter being in association with immunologic reactions that can be minimized by avoiding use in men with > 400 cd4 t - cells/l and women with > 250 cd4 t cells/l . there have been a few case reports of significant hepatotoxicity with the ccr5 antagonist maraviroc , but this has not been clearly seen in the pivotal randomized controlled trials . while there was some increased risk of hepatotoxicity associated with treatment interruption in the smart study , there are currently much fewer data linking ongoing hiv-1 replication to liver abnormalities than what has been described for cardiovascular and renal disease . numerous factors are known to be associated with increased risk of atherosclerotic disease , such as diabetes mellitus , hypertension , smoking , family history and dyslipidemia , all of which occur with variable frequency in those with hiv-1 infection . as combination arv therapy allowed hiv-1-infected individuals to live longer , moreover , concerns are enhanced by the association between select arv agents and insulin resistance , dyslipidemia and fat maldistribution , along with case reports of premature cardiovascular disease . while there is little evidence clearly demonstrating an association between any specific drug and visceral adiposity , lipoatrophy does appear to be a hiv-1-specific condition primarily linked to the use of thymidine analogues such as zidovudine and stavudine . similarly , it is now clear that lipid abnormalities have been seen with select nrtis , non - nrtis , and pis . in fact , there are specific guidelines for the management of dyslipidemia in hiv-1-infected individuals . while several studies have reported a relationship between arv use and cardiovascular events , the d : a : d ( data collection on adverse events of anti - hiv drugs ) cohort is the largest longitudinal study specifically designed to address this question . this study has provided the best evidence that there is an independent relationship between duration of combination arv therapy and cardiovascular events , with most of the effect initially being linked to the use of pis . more recent analyses of this cohort have addressed the relationship between nrti use and cardiovascular disease . here they showed that the recent use of abacavir and , to a lesser extent , didanosine was associated with increased risk of cardiac events when compared to regimens that did not use these drugs . notably the absolute risk was small for all groups and was most prominent amongst those with other cardiovascular risk factors . a follow - up analysis presented at the 16 conference on retroviruses and opportunistic infections showed that there was also an independent association between cardiovascular events and the use of lopinavir / ritonavir and indinavir , while no such relationship was seen with tenofovir disoproxil fumarate ( tenofovir df ) or the non - nrtis efavirenz and nevirapine . it is important to note that while cohorts can include large numbers of longitudinally followed patients , they are not randomized controlled trials and therefore have numerous limitations . that being said , a similar association between abacavir use and cardiovascular events was recently reported from the smart ( strategies for management of anti - retroviral therapy ) study and a case control study of patients in the anrs ( national agency for aids research ) french hospital cohort . nevertheless , questions remain since there has been no definitive biological explanation given for these findings and other studies have not demonstrated such associations . intriguing data have recently emerged to suggest a relationship between ongoing viremia and cardiovascular disease . data from the smart study , designed to assess whether treatment interruption in those with higher levels of cd4 t cells could minimize some of the toxicity associated with arv therapy , actually showed overall increased risk of several non - aids events , including cardiovascular , renal and hepatic disease , amongst those that interrupted treatment . further analyses from the smart study have demonstrated that markers of inflammation , such as interleukin-6 ( il-6 ) , and coagulation , such as d - dimers , are increased in those off arv therapy , all of these markers having been shown in other populations to be associated with increased risk of cardiovascular events . stored samples from the smart study have further been used to link these changes in markers of inflammation with abacavir use . in a cross - sectional analysis of patients either on or off abacavir at baseline , the investigators showed increased levels of high sensitivity c - reactive protein and il-6 in those on abacavir compared to those not taking this drug . however , another study showed different results , demonstrating that related markers declined to a similar extent in both arms in a randomized controlled trial comparing tenofovir df / emtricitabine to abacavir / lamivudine . these differences may reflect the difficulty in observing a potentially modest relationship between select arv agents and markers of inflammation in the face of the overwhelming influence that suppressing plasma hiv-1 rna has on these same measures . while results are inconclusive , other potential markers associated with increased cardiovascular disease have also been evaluated as explanations for the possible relationship between select arv agents and cardiovascular events , such as endothelial function and platelet aggregation . while there remains considerable uncertainty as to how these various data sets should be interpreted , these observations have led to new research exploring the relationship between hiv-1 replication and potential pathogenic mechanisms for select end - organ events . renal disease is common amongst those with hiv-1 infection and is often multifactorial ; possible factors include hiv-1-associated nephropathy and co - morbid conditions such as diabetes mellitus and hypertension , as well as co - infection with hepatitis b and c . certain arv drugs have also been associated with nephrotoxicity , such as indinavir , a drug rarely used in the current era that frequently caused nephrolithiasis and occasionally interstitial nephritis . in addition , tenofovir df has been linked to the development of proximal renal tubular dysfunction . it is clearly recommended that routine monitoring of renal function should occur in all hiv-1-infected patients , with particular attention given to those with co - morbid conditions or taking nephrotoxic drugs . one recent study attempted to define the relationship between renal function , tenofovir df use , and the degree of plasma hiv-1 rna suppression . this was a relatively small cohort study showing that those with complete viral suppression on a tenofovir df - containing regimen actually experienced an increase in glomerular filtration rate . in contrast , there was a small but significant decline in renal function amongst those on tenofovir df that did not achieve full virologic suppression . the authors hypothesize that ongoing viremia , and perhaps the associated increase in inflammation , could be contributing to these adverse outcomes . other studies have shown a similar relationship between hiv-1 replication and progression of renal disease . the overwhelming burden of hepatic disease in hiv-1-infected individuals is related to co - infection with hepatitis b and c . hiv-1 and hepatitis co - infection treatment guidelines provide detailed information about relevant interactions between these chronic viral infections and how co - infection influences the management of each pathogen . there is also an increased risk of hepatic steatosis that may be associated with hyperlipidemia , insulin resistance and select arv agents . several arvs have also been shown to result in hepatotoxicity , the strongest links being with high - dose ritonavir , rarely used in the current treatment era , and tipranavir and nevirapine , the latter being in association with immunologic reactions that can be minimized by avoiding use in men with > 400 cd4 t - cells/l and women with > 250 cd4 t cells/l . there have been a few case reports of significant hepatotoxicity with the ccr5 antagonist maraviroc , but this has not been clearly seen in the pivotal randomized controlled trials . while there was some increased risk of hepatotoxicity associated with treatment interruption in the smart study , there are currently much fewer data linking ongoing hiv-1 replication to liver abnormalities than what has been described for cardiovascular and renal disease . a thorough understanding of the relationship between different drugs and various adverse events is critical to the optimal management of such patients . the first step towards safely using any medication is to know what conditions any given individual is predisposed to , to understand the safety profile of each drug , and to monitor for adverse events . in the case of cardiovascular disease it is important to emphasize efforts to modify known risk factors and to monitor and manage dyslipidemia . when lipid abnormalities are present , clinicians should be aware of how different medications may be contributing to these problems and should consider changes in therapy when appropriate . the emerging data linking select drugs to cardiovascular disease , while not definitive , should also be considered when making any clinical decision , particularly in those with other risk factors . similar considerations apply to underlying renal disease , where control of traditional risk factors such as diabetes mellitus and hypertension should be prioritized along with careful monitoring and avoidance of nephrotoxic drugs in those at greatest risk . with regards to liver disease , the best strategy is to diligently screen for hepatitis b and c co - infection , minimize use of hepatotoxic agents and to provide immunization against viral hepatitis when appropriate . in addition , all patients should be carefully monitored for liver disease , with a particular focus on those taking hepatotoxic agents and who are hepatitis b and/or c co - infected . the recent data linking ongoing hiv-1 replication with cardiovascular disease , and possibly renal disease , are provocative , and the association remains an area of increasing investigation . clinicians should be aware of these studies , their potential implications with regards to the use of arv therapy , and how these findings might support the earlier initiation of treatment in a given individual . however , this needs to be balanced by the fact that the studies remain preliminary and the results thus far have been mostly hypothesis generating . furthermore , decisions regarding the timing of treatment must be made in the context of the specific patient to be treated as well as the overall costs and known risks associated with the use of arv therapy . he is a scientific consultant for abbott , bristol myers squibb , gilead , glaxosmithkline , merck , pfizer , schering plough , pathway , tibotec and serves on a data safety monitoring board for ardea biosciences .

introduction : vitamin d has a basic role in bone growth and metabolism and has been noticed for its important role in many diseases , such as diabetes , depression , hypertension , and cardiovascular disease . regarding some studies , detection of vitamin d deficiency in different places has important implication for health . this study determined prevalence of vitamin d deficiency in arak , a centrally located city in iran.materials and methods : based upon a cross - sectional study in 2010 , 420 students 10 - -16 years old including 220 girls and 200 boys , studied at arak secondary schools , were selected by a multistage sampling . the level of 25 ( oh ) d and pth ( parathormone ) was measured and also the prevalence of vitamin d deficiency in different intensities was evaluated and compared between girls and boys by the student t - test . vitamin d deficiency for the students was categorized into three intensities based on three levels of 25 ( oh ) d : mild deficiency - 15 25 ( oh ) d < 20 ng / ml , moderate deficiency -- 8 25 ( oh ) < 15 ng / ml , severe deficiency - 25 ( oh ) < 8 ng / ml . the relationship between the 25 ( oh ) d and pth was assessed by scatter chart to define cut - off points for vitamin d deficiency.results:based on 25 ( oh ) d < 20 ng / ml , 84% of the students had vitamin d deficiency including 60.7% mild , 22.4% moderate , and 0.7% severe . three local cut - off points including 13 , 21 , and 33 ng / ml were defined in the scatter chart . the results showed that the deficiency of vitamin d in girls is much more than that in boys ( p = 0.05).conclusion : the high prevalence of vitamin d deficiency could be probably a result of different etiologies . we recommended that people should be encouraged to take vitamin d and calcium supplements and also they should have more exposure to the sunlight . the first 15 years of the human immunodeficiency virus type 1 epidemic was characterized by patients progressing to clinical acquired immunodeficiency syndrome and death . the availability of potent antiretrovirals led to the recognition of unique adverse events associated with select drugs . more recent data suggest that end - organ damage may be associated with ongoing viremia . further understanding of the potential role different drugs and the virus itself has on various organs can enhance the clinician 's ability to manage patients in the clinic .

surgical instruments and materials continue to be retained in the peritoneal cavity despite precautionary measures . even though uncommon it is also under - reported and carries serious medico - legal consequences . gauzes and sponges ( gossypiboma ) are the most commonly retained materials and intra - abdominal retained artery forceps are much rarer but when they do occur lead to chronic abdominal pain and can be a rare cause of intestinal obstruction or strangulation with significant morbidity and mortality . we present a case of intraabdominal retained artery forceps in a 70-years - old lady who underwent laparotomy with splenectomy for a large spleen in a peripheral hospital . upon discharge 12 months later she presented to us with an acute ( surgical ) abdomen requiring another laparotomy . at laparotomy she had strangulated / gangrenous lower jejunual and upper ileal bowel loops , the small bowel mesentery of this area being tightly trapped between the jaws of the retained artery forceps retained instruments in intra - abdominal surgery can cause serious complication and should be treated surgically . high index of suspicion and appropriate investigations like plain abdominal x - ray , abdominal ultrasound and ct and mri scans should be instituted in patients who develop chronic abdominal symptoms following laparotomy . preventive measures against retained instruments the rarity of reported cases may be due to medico - legal implications or the surgeon 's unwillingness to advertise his errors . the incidence of retained foreign body in literature is 1 per 1000 to 1500 laparotomies . locally here , the incidence is not documented but it is suspected that the incidence is of a higher magnitude bearing in mind the level of socio - economic development in our environment . with increasing awareness of the public to their rights and privileges , this medico - legal problem will in the near future retained foreign body in peritoneal cavity is one of the known but avoidable complications of abdominal surgery . it is more liable to occur when surgery is done in a non - ideal environment and negligence or ignorance on part of the medical personnel . it is the responsibility of the operating surgeon and the theatre staff to ensure that no foreign body is left inside the peritoneal cavity before closure of the abdominal cavity . retained foreign bodies are of various types and include sponges / gauzes ( gossypiboma or texilloma ) , artery forceps , other metal instruments , surgical needles , surgical drains , and rubber tubes . the surgical sponge / gauze ( gossiypiboma ) constitutes the most frequently encountered object because of its common usage , small size and amorphous structure . exudative reaction will give rise to abscess formation resulting in peritonitis , fistula formation , tender abdominal mass and gut perforation . in contrast retained artery forceps remain inert and induce their effects through mechanical means giving rise to pressure effects resulting in abdominal pain or direct tissue / viscus injury leading to tissue strangulation or perforation of viscus leading to for example peritonitis or vascular injury resulting in hemorrhage . the clinical presentation of retained intra - abdominal foreign material may be acute or delayed . such patient present in the surgical units with increasing abdominal pain , abdominal mass , discharging sinus , intra - abdominal abscesses and acute or sub - acute intestinal obstruction . whilst many patients will present within days or few weeks after the initial surgery and have a re - laparotomy for retrieval of the foreign body , some patients with intra - abdominal foreign material may go unnoticed for years or even decades . however many of this subset of patients will have chronic abdominal symptoms such as abdominal pain or discomfort . the pre - operative diagnosis is based on history , clinical examination , plain abdominal radiographs , ultrasound , ct and mri scans . yet generalized peritonitis , systemic complications and presentation of acute intestinal obstruction are associated with high mortality . we present a case of retained artery forceps causing intestinal strangulation managed in our centre and review the possible factors that led to the retained artery forceps and advice on possible preventive measures . a 70yr old woman ( e.n ) presented to our accident and emergency unit as a referral from a cottage ( peripheral ) hospital with a complaint of sudden exacerbation of her chronic abdominal pain , associated now with abdominal distension , vomiting , absolute constipation and fever of four days duration . 12 months prior to presentation she had laparotomy with splenectomy for large spleen suspected to be due to tropical splenomegaly syndrome ( tss ) . few weeks following the splenectomy , she started complaining of intermittent abdominal pains of worsening severity which culminated in her present complaint . she had paid severe visits to the surgeon who did her splenectomy and had analgesis and antispasmodics prescribed and was always reassured that the abdominal pain will abate with time . examination revealed an acutely ill looking , toxic , pale and dehydrated elderly lady in respiratory distress , blood pressure of 80/50mmhg , pulse rate of 120/min and low volume , respiratory rate 40/min and a temperature of 38.4c . abdominal examination revealed a distended abdomen with a midline scar from the epigastrum to the symphysis pubis . chest examination revealed severe dyspnoea and bilateral basal rales . a clinical diagnosis of generalized peritonitis complicating intestinal obstruction , septic shock and acute respiratory distress syndrome ( ards ) were made . resuscitation was started with iv saline infusions , iv ceftriazone ( third generation cephalosporins ) , iv metronidazole , nasogastric tube suction , and urethral catheterization ( for urinary output monitoring ) . investigation showed packed cell volume of 22% , urea of 11.5 mmol / l , normal electrolytes and urinalysis . plain chest and abdominal ( erect and supine ) x - ray were ordered but were not done because the poor state of patient prevented her movement to the x - ray suit . however , she had abdominal pelvic ultrasound , which showed dilated bowel loops and large volume of intraperitoneal fluid . the jaws of a large size artery forceps was seen tightly trapping the small bowel mesentery from its distal extremity to half way up towards the duodeno - jejunal flexure with the tips of forceps pointing toward the duodeno - jejunal junction and the handle located in the right iliac fossa . this led to ischemic gangrene of about 65 cm of the small bowel involving the distal third of jejunum and upper two thirds of the ileum . there were no pulsations on the ileal and jejunal arteries distal to the trapping artery forceps ( figures 1 and 2 ) . the jaws of the artery is seen trapping the small bowel mesentery with the handle in the right iliac fossa and the tip towards the duodeno - jejunal junction . loops of gangrenous small bowel are held up by an assistant above the artery forceps showing artery forceps across small bowel mesentery . the patient had a turbulent post- operative phase and was managed in our critical care unit ( ccu ) . however , 12-hours post - operatively she died most probably from continuing sepsis . despite the awareness of the dangers of and the precautionary measures aimed to prevent them retained instruments continue to occur especially in the peritoneal cavity . retained foreign body in a patient after surgery is an uncommon but dangerous error and it is grossly under estimated . the rarity of the reported cases may be due to medico - legal reasons or the surgeon 's unwillingness to advertise his errors or complacency of his colleagues in exposing him , in order not to jeopardize his professional life or to protect him from medico - legal issues that might ensure . in literature the incidence of retained foreign body is quoted as 1 : per 1000 to 1500 laparotomies . here in our environment however , given the level of our socio - economic and manpower development in health care , it is strongly suggested that the incidence in our environment is much high than quoted in the literature . in our environment people are gradually beginning to appreciate their rights and privileges and the authors suspect that in the years ahead the medico legal issues associated with retained foreign bodies following surgery will be in the front burner . the most common retained foreign body is abdominal sponge ( gossypiboma ) because of its common usage and amorphous structure . whilst gossypiboma elicits two types of responses - asceptic fibrinous and exudative reaction , retained metals like artery forceps are inert . while many retained foreign materials are identified and retrieved immediately or shortly after surgery some may remain unidentified for years or decades . qamar , champ and nasir found one case each of retained artery forceps in their reviews of eleven , fifteen and ten cases respectively of retained foreign bodies in the abdominal cavity . retained metal like artery forceps are inert and exert their effect through mechanical means like direct tissue damage causing ischemia , pressure effect giving rise to pain and discomfort , or forming an axis on which loops of bowel entangle leading to intestinal obstruction and strangulation or a loop of bowel going through the eye of the retained artery forceps and getting trapped leading to strangulation . they may also penetrate hollow organs for example gut leading to peritonitis or blood vessels leading to hemorrhage . in our case , the artery forceps through its clinching jaws caused ischemia ( direct tissue damage ) leading to strangulation and gangrene of small bowel loops . metal retained materials are said to usually give rise to acute symptoms months or years after the operation . fadiora and godara noted in their cases that their patients presented with acute symptoms after 2 years and 2 years respectively of their initial surgical operations . our patient presented 12 months after surgery . however , in all cases of retained artery forceps in the abdominal cavity sited in literature , the patients had chronic recurrent abdominal pains or colic 's months or years before onset of acute ( surgical ) abdomen . our patient had months of recurrent abdominal pain before the acute ( surgical ) abdomen . in the literature most of the reported cases of retained artery forceps were operated in the peripheral hospitals . our patient had surgery at which retained forceps occurred in a peripheral hospital . in most of the peripheral hospitals in developing country like ours , qualified medical personnel are few and auxiliaries most times inexperienced are called in to help in the operating theatre . therefore , any surgeon working in a peripheral hospital who decides to undertake any major surgery must assume full responsibility of preventing retained instruments and must involve his assistants and auxiliaries in his anxiety . also in most of hospital theatres , adequate equipment and facilities are lacking , improvisation is key to many procedures and with this disasters are common including retained instruments . a recent retrospective study found that retained instruments and sponges following an operation occurred more frequently in emergency surgery , especially where there was an unplanned change in the operative procedure , in patients with high mean body mass index . others noted that huge intra - abdominal tumors or mass and severe intra- operative hemorrhage are risk factors for retained instrument . other risk factors are fatigued surgical team , staff changes during operation , and failure to account for all sponges and instruments , leaving closure of abdominal wound to a junior , sometimes inexperienced . the surgeon remains the head of the surgical team and must assume full responsibility for what he puts on the patient even though the general custom and practice is to delegate the task of counting sponges and other instruments to the nurses . the responsibility of the surgeon is even more important and pertinent in peripheral hospitals where trained peri - operative nurses are very scarce , and the surgeon uses nurse auxiliaries- who are most times untrained the following precautionary measures are likely to reduce the incidence of retained instruments : double sponge / instrument count , before and after surgery.number of sponges and instruments are clearly written in the theatre black board provided for this purpose.all large sponges ( abdominal mops ) applied into the peritoneal cavity should have long ribbon attached to the sponge and the ribbon must remain outside the main laparotomy wound.avoid use of small gauze inside the abdominal cavity ; only use large abdominal mops ( sponges with long ribbon attached).the surgeon must develop the attitude of thorough final sweep(visual and manual ) of the abdominal cavity to ensure no instruments or sponges are left inside the abdominal cavity before commencement of the wound closure.uses of sponges with radio - opaque markers are advised but these materials are not easily available to us here . also advocated is radiographic screening of high risk operative procedures before patients leave the operating room , even when counts are documented as correct . number of sponges and instruments are clearly written in the theatre black board provided for this purpose . all large sponges ( abdominal mops ) applied into the peritoneal cavity should have long ribbon attached to the sponge and the ribbon must remain outside the main laparotomy wound . avoid use of small gauze inside the abdominal cavity ; only use large abdominal mops ( sponges with long ribbon attached ) . the surgeon must develop the attitude of thorough final sweep(visual and manual ) of the abdominal cavity to ensure no instruments or sponges are left inside the abdominal cavity before commencement of the wound closure . uses of sponges with radio - opaque markers are advised but these materials are not easily available to us here . also advocated is radiographic screening of high risk operative procedures before patients leave the operating room , even when counts are documented as correct . this is ideal but not feasible in a developing world setting . in our environment and especially for surgeon operating in peripheral hospitals where the incidence of retained materials are much more commoner , he must rely on his own ability of making absolutely sure that all the procedures adopted for prevention of retained materials in wounds are carried out . no reliance should be placed on other theatre staffing since the short comings of these staff are obvious . these measures are even more pertinent when operating on a high risk patient for retained instruments . exploratory laparotomy remains the mainstay of treatment to retrieve retained intra - abdominal foreign material . other methods include laparoscopic removal and ultrasound assisted retrieval rarely small sponges and gauzes can discharge via the rectum spontaneously . it is advocated that patient who present with chronic abdominal discomfort , pain or colic 's , after laparotomy should be subjected to thorough history taking , clinical examination and should have plain abdominal x - rays , supplemented by ultrasound scan , and perhaps ct scan and possibly mri scan . adoption of this procedure will lead to early diagnosis of retained foreign materials , before the patient develops acute ( surgical ) abdomen . retained foreign body is avoidable and could be a costly error as our case illustrates . the need for the surgeon ( especially the rural surgeon ) who operates in the abdominal cavity to assume the captain of ship doctrine can not be over emphasized . all preventive protocols must be observed under the strict guidance and full responsibility of the surgeon especially the rural surgeon . the need to investigate properly a patient who complains of chronic abdominal symptoms following laparotomy should be encouraged as this will aid in making early diagnosis of retained foreign material before more serious complications of intestinal obstruction or strangulation , or peritonitis , intra - abdominal abscesses or entero - cutanous fistula supervenes . like many developed nations and even some developing ones , jamaica is able to boast of its notable achievement in progress made toward advancing the health status of its populace , during the twentieth century which are expressed in the postponement of death , lowered fertility , high nutrition and sanitation and more importantly the increasing life expectancy . life expectancy which is an indicator of health status revealed that the country 's health status is reasonably good , as the life span for jamaicans was similar to those in some first world societies interestingly , the biological science highlighted that the ageing process comes with changes in physical functioning . according to the merck manual of health and aging older cells function less well . also , in some organs , cells die and are not replaced , so the number of cells decreases , indicates not only the decline functionality of aged body but also the role of diseases in this regards . the oldest - old categorization is said to be the least physical functioning compared to the other classification in chronological ageing . the young - old , on the other hand , are more likely to be the most functioning as the organism is just beginning the transition into the aged arena . a study conducted by costa , using secondary data drawn from the records of the union army ( ua ) pension programme that covered some 85% of all ua , showed that there was an association between chronic conditions and functional limitation which include difficulty walking , bending , blindness in at least one eye and deafness . those functional activities are classified as adl ( activities of daily living ) or ( i ) adl ( instrumental activities for daily living ) . these include activities such as feeding oneself ; bathing , dressing , grooming , work , homemaking and leisure . the ( i ) adl are those activities whose accomplishment is necessary for continued independent residence in the community . the independent activities of daily living are more sensitive to subtle functional deficiencies than adl 's and differentiate among task performance including the amount of help needed to accomplish each task . within the context of ageing and the reality of having chronic diseases , ones adl and ( i ) adl will be hampered somewhat . some illnesses , such as huntington 's disease , hypertension , heart diseases , diabetes mellitus , cancer , cataract , and stroke , result in a gradual loss of the ability to provide self - care and some result in an immediate dependence or lowered functional capacity and sometimes even mortality . hence , if ageing were associated with no ailments , it still comes with reduced functional capacity . according to eldemire the majority of jamaican older persons are physically and mentally well and living in family units , suggesting that illnesses are eroding some of functional capacity of elderly jamaica which is synonymous to ageing . despite eldemire 's findings , a study on the elderly published in the caribbean food and nutrition institute 's magazine ( i.e. cajanus ) found that 70% of individuals who were patients within different typologies of health services were senior citizens , suggesting that elderly jamaicans were not only spending more time utilizing health care services than other age cohorts but that they were experiencing lowered functional capacity . the aforementioned health literature has shown that diseases positively influence functionality , and kim et al . they found that mci ( i.e. mild cognitive impairment ) patients performed significantly worse on four out of a total 15 items ( i.e. telephone , transportation , finances and household appliances ) of elderly 60 + year old . another descriptive study conducted by natividad and zimmer when further than kim et al . in 2000 , they found that 11.5% were having difficulties walking in the house ; 8.0% difficulty bathing ; 6.3% dressing themselves ; and 4.6% eating . on the issue of ( i)adl for natividad and zimmer 's work , for 2000 , 18.5% had difficulty with using transportation ; 17.6% shopping ; 13.8% preparing meals ; 13.8% light housework ; and 9.4% manage their money . in the same aforementioned study , using logistic regression , they went further and found that age was the significant factor that determine adl ( or=1.08 , p < 0.05 ) ; while age ( or = 1.07 , p < 0.05 ) and area of residence ( i.e. rural or=0.66 , furthermore , they found that marital status , education , and gender were not statistical significant determinants of adl or ( i ) adl . this study is timely as it aimed to examine adl , ( i ) adl and sought to investigate those determinants of functional capacity of older men in jamaica . using data for 2007 on 2,000 jamaican men 55 + years , the current study evaluated activities of daily living ( adl ) , instrumental activities ( ( i)adl ) , self - reported health status and using logistic regression , determined those factors that explain functional capacity of older men . the current study therefore will not only provide information upon which public health policies can be fashioned ; but it also will aid an understanding of older men and how they perceived health and determined their adl , ( i ) adl and those factors that influence their functional abilities and not assume that what obtains in other locality equally applies to jamaican men 55 + years . the study used primary cross - sectional survey data on men 55 years and older from the parish of st . the survey was submitted and approved by the university of the west indies medical faculty 's ethics committee . stratified multistage probability sampling technique was used to draw the sample ( 2,000 respondents ) . the instrument was sub - divided into general demographic profile of the sample ; past and current health status ; health - seeking behavior ; retirement status ; social and functional status . data was stored , retrieved and analyzed , using spss for windows ( 16.0 ) ( spss inc ; chicago , il , usa ) . the statistical institute of jamaica ( statin ) maintains a list of enumeration districts ( ed ) or census tracts . catherine is divided into a number of constituencies made up of a number of enumeration districts ( ed ) . the enumeration districts were listed and numbered sequentially and selection of clusters was arrived by the use of a sampling interval . forty enumeration districts ( clusters ) were subsequently selected with the probability of selection being proportional to population size ( table 1 ) . proportion of survey ( sample ) vs. proportion of population the sample population does not only speak to the parish of st . the sampling frame was men fifty - five years and older in the parish of st catherine . catherine was chosen as previous data and surveys[1113 ] suggested that it has the mix of demographic characteristics ( urban , rural and age - composition ) which typify jamaica . for the current study descriptive status employed to provide background information on the sample ; and chi - square was used to examine non - metric variables . level of significance was p - value < 0.05 and the only exclusion criterion was if more than 20% of the cases of a variable were missing . this variable is measured using people 's self - rate of their overall health status , which ranges from excellent to poor health status . the question was how would you rate your health today? ( 1 ) excellent ; ( 2 ) good ; ( 3 ) fair and ( 4 ) poor . what is [ your ] highest level of [ education ] attained ? the options were ( 1 ) no formal education ; ( 2 ) basic school ; ( 3 ) primary school / all age ; ( 4 ) secondary / high / technical school ; ( 5 ) vocational ( i.e. apprenticeship / trade ) ; ( 6 ) diploma ; ( 7 ) undergraduate degree ; ( 8) post - graduate degree do you take time out for regular exercise? ( 1 ) yes and ( 2 ) no . type of physical exercise . ( 1 ) yes , ( 2 ) no . if the response to either question was yes , this was coded as poor childhood health status and if the response was no in both cases it was coded a good health status in childhood . these are ( 1 ) ages 55 to 64 years ; ( 2 ) ages 65 to 74 years ; and ( 3 ) ages 75 years and older ( i.e. 75 + years ) . performance of activities of daily living ( adl ) is used to describe the functional status of a person . it is used to determine a baseline level of functioning and to monitor improvement in activities of daily living ( adl ) overtime . scoring the adl findings ( katz ) independence on a given function received a score of 1 point while if dependent , 0 point was given . there were 6 items ( eating refers to feeding oneself ; dressing denotes getting clothes and getting dressed , including typing shoes ; transferring means to get in and out of bed as well as in and out of a chair ; using toilet refers to going to the toilet and cleaning afterwards ; bathing denotes to sponge bath , shower , tub bath , or washing body with a wet towel ; continence denotes to control of urination and bowel movement ) . total scores thus could range from 0 to 6 with lower scores indicating low independence ( i.e. high dependence ) and larger scores indicating higher independence . if there was a score of 0 to 2 ( i.e. none to 2 of the six adl activities was chosen ) , the older person was classified as low independence ; if 3 to 4 of the activities were selected , the older man was classified as moderately independent and if 5 to 6 items were selected the older was classified as highly independent . instrumental activities of daily living ( ( i ) adl ) . the instrumental activities of daily living tool was the basis for assessing participants difficulty with ( i ) adl . ( i)adl are those activities whose accomplishment is necessary for continued independent residence in the community . the independent activities of daily living are more sensitive to subtle functional deficiencies than adl 's and differentiate among task performance including the amount of help needed to accomplish each task . hence , ( i ) adl for older men in this study used the 8-item choices as is used for women . these are preparing meals ; shopping ; management medication ; money management ; transportation ; telephone and laundry . scoring the ( i ) adl . ( i)adl scores reflect the number of areas of impairment i.e. the number of skills / domains in which subjects are dependent . scores range from 0 to 8 , with higher scores indicating higher dependence and lower scores greater independence ( i.e. low dependence ) . if none to 3 activities were selected , the older person was classified as high dependence ; if 4 to 6 activities were selected the elder was classified as moderately dependent and if 7 to 8 items were selected the elder was classified as highly dependent . the cronbach alpha for the 8 item scales was 0.648 . in order to examine the effect of many variables on a single dependent variable , the researcher used multivariate analysis to test a single hypothesis ( physical functioning is determined by current health status , happiness , area of residence see equation . natividad and zimmer had used logistic regression to examine factors that determined adl , ( i ) adl and self - reported of older filipinos . using the literature , the current study investigated the correlates of functional status of older jamaicans within the context of the available data . the proposed model that this research seeks to evaluate is displayed ( eqn1 ) : fi = 0 + 1hti + 2happi + 3lsi + 4chi + 5ari + 6ai + 7ssi + 8cai + 9edi + 10hhi + 11msi + 12pi + 13hai + 14tmi + 15di + i where fi ( physical functionality ) is a function of some current health status , ht ; happiness , happi ; life satisfaction , lsi ; children , ci ; area of residence , ari ; age group of respondent , ai ; social support , ssi ; church attendance , cai ; educational level , edi ; head of household , hhi ; marital status , msi ; number of person in household , pi ; health status in childhood , hai ; employment status , emi ; depression , di ; taking medication , tmi ; health advise , hai . all the variables were identified from the literature . using the principle of parsimony , only those explanatory variables that are statistically significant ( p < 0.05 ) were used in the final model to determine f1 of older men in jamaica . this final model identified the correlates of fi of older men in jamaica , ( eqn2 ) . fi = 0 + 1ari + 2ai + 3ssi + 4cai + 5pii furthermore , the variables used in this study are based on ( 1 ) literature review which shows that these are likely to correlate with the particular dependent variable , and 2 ) the correlation matrix was examined in order to ascertain if autocorrelation ( or multicollinearity ) existed between independent variables . based on bryman and cramer , correlation can be low ( weak ) from 0 to 0.39 ; moderate any of the independent variables which had moderate to high correlation was excluded from the model . the correlation between life satisfaction and happiness was 0.633 ; happiness and social networking ( correlation coefficient = 0.12 , p = 0.003 ) ; happiness and marital status ( correlation coefficient = 0.107 , p = 0.026 ) ; marital status and income category ( correlation coefficient = 0.193 , p < 0.001 ) ; social networking and marital status ( r=0.205 , p < 0.001 ) ; social networking and age group ( correlation coefficient = 0.188 , p < 0.001 ) ; social networking and occupation ( correlation coefficient = 0.320 , p < 0.001 ) ; social networking educational category ( correlation coefficient = 0.420 , p < 0.001 ) ; adl and age cohort ( correlation coefficient = -0.813 , p = 0.032 ) ; income and occupation ( correlation coefficient = 0.7775 , p < 0.001 ) ; and , income and education ( correlation coefficient = 0.356 , p<0.001 ) ; employment and education category ( correlation coefficient = 0.283 , p < 0.001 ) , and depression and life satisfaction ( correlation coefficient = 0.160 , p < 0.001 ) . however , there was no correlation between happiness and present occupation ( p = 0.761 ) ; happiness and income ( p = 0.233 ) ; happiness and employment status ( p = 0.516 ) ; health status and depression ( p=0.876 ) as well as life satisfaction and employment status ( p = 0.261 ) . hence , life satisfaction and happiness ; occupation and income category will not be simultaneously used as explanatory variables . this variable is measured using people 's self - rate of their overall health status , which ranges from excellent to poor health status . the question was how would you rate your health today? ( 1 ) excellent ; ( 2 ) good ; ( 3 ) fair and ( 4 ) poor . what is [ your ] highest level of [ education ] attained ? the options were ( 1 ) no formal education ; ( 2 ) basic school ; ( 3 ) primary school / all age ; ( 4 ) secondary / high / technical school ; ( 5 ) vocational ( i.e. apprenticeship / trade ) ; ( 6 ) diploma ; ( 7 ) undergraduate degree ; ( 8) post - graduate degree do you take time out for regular exercise? ( 1 ) yes and ( 2 ) no . type of physical exercise . ( 1 ) yes , ( 2 ) no . and , were you frequently ill as a child ? ( 1 ) yes , ( 2 ) no . if the response to either question was yes , this was coded as poor childhood health status and if the response was no in both cases it was coded a good health status in childhood . these are ( 1 ) ages 55 to 64 years ; ( 2 ) ages 65 to 74 years ; and ( 3 ) ages 75 years and older ( i.e. 75 + years ) . performance of activities of daily living ( adl ) is used to describe the functional status of a person . it is used to determine a baseline level of functioning and to monitor improvement in activities of daily living ( adl ) overtime . scoring the adl findings ( katz ) independence on a given function received a score of 1 point while if dependent , 0 point was given . there were 6 items ( eating refers to feeding oneself ; dressing denotes getting clothes and getting dressed , including typing shoes ; transferring means to get in and out of bed as well as in and out of a chair ; using toilet refers to going to the toilet and cleaning afterwards ; bathing denotes to sponge bath , shower , tub bath , or washing body with a wet towel ; continence denotes to control of urination and bowel movement ) . total scores thus could range from 0 to 6 with lower scores indicating low independence ( i.e. high dependence ) and larger scores indicating higher independence . if there was a score of 0 to 2 ( i.e. none to 2 of the six adl activities was chosen ) , the older person was classified as low independence ; if 3 to 4 of the activities were selected , the older man was classified as moderately independent and if 5 to 6 items were selected the older was classified as highly independent . the instrumental activities of daily living tool was the basis for assessing participants difficulty with ( i ) adl . ( i)adl are those activities whose accomplishment is necessary for continued independent residence in the community . the independent activities of daily living are more sensitive to subtle functional deficiencies than adl 's and differentiate among task performance including the amount of help needed to accomplish each task . hence , ( i ) adl for older men in this study used the 8-item choices as is used for women . these are preparing meals ; shopping ; management medication ; money management ; transportation ; telephone and laundry . scoring the ( i ) adl . ( i)adl scores reflect the number of areas of impairment i.e. the number of skills / domains in which subjects are dependent . scores range from 0 to 8 , with higher scores indicating higher dependence and lower scores greater independence ( i.e. low dependence ) . if none to 3 activities were selected , the older person was classified as high dependence ; if 4 to 6 activities were selected the elder was classified as moderately dependent and if 7 to 8 items were selected the elder was classified as highly dependent . the cronbach alpha for the 8 item scales was 0.648 . in order to examine the effect of many variables on a single dependent variable , the researcher used multivariate analysis to test a single hypothesis ( physical functioning is determined by current health status , happiness , area of residence natividad and zimmer had used logistic regression to examine factors that determined adl , ( i ) adl and self - reported of older filipinos . using the literature , the current study investigated the correlates of functional status of older jamaicans within the context of the available data . the proposed model that this research seeks to evaluate is displayed ( eqn1 ) : fi = 0 + 1hti + 2happi + 3lsi + 4chi + 5ari + 6ai + 7ssi + 8cai + 9edi + 10hhi + 11msi + 12pi + 13hai + 14tmi + 15di + i where fi ( physical functionality ) is a function of some current health status , ht ; happiness , happi ; life satisfaction , lsi ; children , ci ; area of residence , ari ; age group of respondent , ai ; social support , ssi ; church attendance , cai ; educational level , edi ; head of household , hhi ; marital status , msi ; number of person in household , pi ; health status in childhood , hai ; employment status , emi ; depression , di ; taking medication , tmi ; health advise , hai . all the variables were identified from the literature . using the principle of parsimony , only those explanatory variables that are statistically significant ( p < 0.05 ) were used in the final model to determine f1 of older men in jamaica . this final model identified the correlates of fi of older men in jamaica , ( eqn2 ) . fi = 0 + 1ari + 2ai + 3ssi + 4cai + 5pii furthermore , the variables used in this study are based on ( 1 ) literature review which shows that these are likely to correlate with the particular dependent variable , and 2 ) the correlation matrix was examined in order to ascertain if autocorrelation ( or multicollinearity ) existed between independent variables . based on bryman and cramer , correlation can be low ( weak ) from 0 to 0.39 ; moderate any of the independent variables which had moderate to high correlation was excluded from the model . the correlation between life satisfaction and happiness was 0.633 ; happiness and social networking ( correlation coefficient = 0.12 , p = 0.003 ) ; happiness and marital status ( correlation coefficient = 0.107 , p = 0.026 ) ; marital status and income category ( correlation coefficient = 0.193 , p < 0.001 ) ; social networking and marital status ( r=0.205 , p < 0.001 ) ; social networking and age group ( correlation coefficient = 0.188 , p < 0.001 ) ; social networking and occupation ( correlation coefficient = 0.320 , p < 0.001 ) ; social networking educational category ( correlation coefficient = 0.420 , p < 0.001 ) ; adl and age cohort ( correlation coefficient = -0.813 , p = 0.032 ) ; income and occupation ( correlation coefficient = 0.7775 , p < 0.001 ) ; and , income and education ( correlation coefficient = 0.356 , p<0.001 ) ; employment and education category ( correlation coefficient = 0.283 , p < 0.001 ) , and depression and life satisfaction ( correlation coefficient = 0.160 , p < 0.001 ) . however , there was no correlation between happiness and present occupation ( p = 0.761 ) ; happiness and income ( p = 0.233 ) ; happiness and employment status ( p = 0.516 ) ; health status and depression ( p=0.876 ) as well as life satisfaction and employment status ( p = 0.261 ) . hence , life satisfaction and happiness ; occupation and income category will not be simultaneously used as explanatory variables . this variable is measured using people 's self - rate of their overall health status , which ranges from excellent to poor health status . the question was how would you rate your health today? ( 1 ) excellent ; ( 2 ) good ; ( 3 ) fair and ( 4 ) poor . what is [ your ] highest level of [ education ] attained ? the options were ( 1 ) no formal education ; ( 2 ) basic school ; ( 3 ) primary school / all age ; ( 4 ) secondary / high / technical school ; ( 5 ) vocational ( i.e. apprenticeship / trade ) ; ( 6 ) diploma ; ( 7 ) undergraduate degree ; ( 8) post - graduate degree do you take time out for regular exercise? ( 1 ) yes and ( 2 ) no . type of physical exercise . ( 1 ) yes , ( 2 ) no . and , were you frequently ill as a child ? ( 1 ) yes , ( 2 ) no . if the response to either question was yes , this was coded as poor childhood health status and if the response was no in both cases it was coded a good health status in childhood . these are ( 1 ) ages 55 to 64 years ; ( 2 ) ages 65 to 74 years ; and ( 3 ) ages 75 years and older ( i.e. 75 + years ) . performance of activities of daily living ( adl ) is used to describe the functional status of a person . it is used to determine a baseline level of functioning and to monitor improvement in activities of daily living ( adl ) overtime . scoring the adl findings ( katz ) independence on a given function received a score of 1 point while if dependent , 0 point was given . there were 6 items ( eating refers to feeding oneself ; dressing denotes getting clothes and getting dressed , including typing shoes ; transferring means to get in and out of bed as well as in and out of a chair ; using toilet refers to going to the toilet and cleaning afterwards ; bathing denotes to sponge bath , shower , tub bath , or washing body with a wet towel ; continence denotes to control of urination and bowel movement ) . total scores thus could range from 0 to 6 with lower scores indicating low independence ( i.e. high dependence ) and larger scores indicating higher independence . if there was a score of 0 to 2 ( i.e. none to 2 of the six adl activities was chosen ) , the older person was classified as low independence ; if 3 to 4 of the activities were selected , the older man was classified as moderately independent and if 5 to 6 items were selected the older was classified as highly independent . the instrumental activities of daily living tool was the basis for assessing participants difficulty with ( i ) adl . ( i)adl are those activities whose accomplishment is necessary for continued independent residence in the community . the independent activities of daily living are more sensitive to subtle functional deficiencies than adl 's and differentiate among task performance including the amount of help needed to accomplish each task . hence , ( i ) adl for older men in this study used the 8-item choices as is used for women . these are preparing meals ; shopping ; management medication ; money management ; transportation ; telephone and laundry . scoring the ( i ) adl . ( i)adl scores reflect the number of areas of impairment i.e. the number of skills / domains in which subjects are dependent . scores range from 0 to 8 , with higher scores indicating higher dependence and lower scores greater independence ( i.e. low dependence ) . if none to 3 activities were selected , the older person was classified as high dependence ; if 4 to 6 activities were selected the elder was classified as moderately dependent and if 7 to 8 items were selected the elder was classified as highly dependent . in order to examine the effect of many variables on a single dependent variable , the researcher used multivariate analysis to test a single hypothesis ( physical functioning is determined by current health status , happiness , area of residence natividad and zimmer had used logistic regression to examine factors that determined adl , ( i ) adl and self - reported of older filipinos . using the literature , the current study investigated the correlates of functional status of older jamaicans within the context of the available data . the proposed model that this research seeks to evaluate is displayed ( eqn1 ) : fi = 0 + 1hti + 2happi + 3lsi + 4chi + 5ari + 6ai + 7ssi + 8cai + 9edi + 10hhi + 11msi + 12pi + 13hai + 14tmi + 15di + i where fi ( physical functionality ) is a function of some current health status , ht ; happiness , happi ; life satisfaction , lsi ; children , ci ; area of residence , ari ; age group of respondent , ai ; social support , ssi ; church attendance , cai ; educational level , edi ; head of household , hhi ; marital status , msi ; number of person in household , pi ; health status in childhood , hai ; employment status , emi ; depression , di ; taking medication , tmi ; health advise , hai . all the variables were identified from the literature . using the principle of parsimony , only those explanatory variables that are statistically significant ( p < 0.05 ) were used in the final model to determine f1 of older men in jamaica . this final model identified the correlates of fi of older men in jamaica , ( eqn2 ) . fi = 0 + 1ari + 2ai + 3ssi + 4cai + 5pii furthermore , the variables used in this study are based on ( 1 ) literature review which shows that these are likely to correlate with the particular dependent variable , and 2 ) the correlation matrix was examined in order to ascertain if autocorrelation ( or multicollinearity ) existed between independent variables . based on bryman and cramer , correlation can be low ( weak ) from 0 to 0.39 ; moderate any of the independent variables which had moderate to high correlation was excluded from the model . the correlation between life satisfaction and happiness was 0.633 ; happiness and social networking ( correlation coefficient = 0.12 , p = 0.003 ) ; happiness and marital status ( correlation coefficient = 0.107 , p = 0.026 ) ; marital status and income category ( correlation coefficient = 0.193 , p < 0.001 ) ; social networking and marital status ( r=0.205 , p < 0.001 ) ; social networking and age group ( correlation coefficient = 0.188 , p < 0.001 ) ; social networking and occupation ( correlation coefficient = 0.320 , p < 0.001 ) ; social networking educational category ( correlation coefficient = 0.420 , p < 0.001 ) ; adl and age cohort ( correlation coefficient = -0.813 , p = 0.032 ) ; income and occupation ( correlation coefficient = 0.7775 , p < 0.001 ) ; and , income and education ( correlation coefficient = 0.356 , p<0.001 ) ; employment and education category ( correlation coefficient = 0.283 , p < 0.001 ) , and depression and life satisfaction ( correlation coefficient = 0.160 , p < 0.001 ) . however , there was no correlation between happiness and present occupation ( p = 0.761 ) ; happiness and income ( p = 0.233 ) ; happiness and employment status ( p = 0.516 ) ; health status and depression ( p=0.876 ) as well as life satisfaction and employment status ( p = 0.261 ) . hence , life satisfaction and happiness ; occupation and income category will not be simultaneously used as explanatory variables . most of the sample was lowly dependent ( 77.1% ) ; 55.4% reported a moderate health status and 63.6% indicated satisfied with life sometimes ( table 2 ) . demographic characteristics of sample when functional capacity was disaggregated into adl and ( i ) adl , the following disparities were observed in the findings . of the sample , however , with regards to ( i ) adl , 1.9% had low , 18.6% moderate and 79.6% high scores . of the sample , 43.1% reported that they were suffering from depression compared to 56.9% who stated no to the question of being depressed in the survey period . on examining depression and age cohort , no significant statistical association was between both variables ( p = 0.102 ) . one half of the sample indicated that they spent ja.$100 ( us $ 1.45 ) monthly for medical expenditure ; 34% of the respondents bought their prescribed medication ; 17.1% reported that they have been hospitalized since their sixth birthday and 65.8% reported that they took no medication . of those who mentioned that they were ill during childhood ( 17.5% , n=350 ) , 34.9% said that the illness was measles or chicken pox , 26.3% mentioned asthma , 10.0% pneumonic fever , 8.9% polio , 6.6% accident , 4.6% jaundice , 1.7% hernia , and 5.1% indicated gastroenteritis . twenty four percent of elderly men indicated that they were rarely happy , 40.5% said sometimes , 31.0% mentioned often and only 4.5% reported always . furthermore , 17.7% of the sample reported that they were seriously ill as children . the findings revealed that no statistical correlation existed between adl and age cohort of the sample ( p=0.205 ) . however , a relationship was found between ( i ) adl and age group of the sample ( 2 ( df = 4 ) = 16.011 ; p=0.003 ) ( table 3 ) . on further examination , it was revealed that as an older male increases in age from 55 - 64 years to 65 - 74 years and 75 + years , his high independence falls and while his moderate dependence increases . of those who were 55 - 64 years , 83.0% of them high independence compared to 78.9% of those 65 - 74 years and 73.9% for those 75 + years ( table 3 ) . adl and ( i ) adl by age group of the sample , 41.8% of older men were health literate or have been advised on medical relates conditions , causes , prognosis and precautions compared to 58.3% who were not aware or have been advice by a health care practitioners ( include a pharmacists , community aide ; nurse , or medical technologists ) . the study revealed that no statistical correlation was found between functional capacity of older men in jamaica and health advice ( or health literacy ) p = 0.845 . however , a weak statistical relationship existed between educational level and health literacy ( 2 ( df = 1 ) = 110.165 , p < 0.001 , correlation coefficient = 0.235 ) ( table 4 ) . health literacy by high level of education attained table 5 revealed that adl for men 55 + years was very high for each activity , with 88.5% for continence being the lowest level of independence . for ( i)adl , 56.7% of sample was still able to perform heavy duty housework , 62.7% were still performing their laundry , 98.1% managed their money and 77.8% were still shopping , and 70.2% prepared their own meals . disaggregating adl and ( i ) adl of sample no statistical correlation was found between health status and age cohort of respondents ( p=0.051 ) , and between life satisfaction and age cohort ( p = 0.430 ) as well as health status and area of residence ( p = 0.190 ) . a significant statistical difference between life satisfaction of urban and rural older men in jamaica ( df = 3 ) = 13.910 , p = 0.003 . however that the correlation was a weak one ( correlation coefficient = 0.083 ) . on further examination , 35.9% of older rural men revealed that they were rarely satisfied with life compared to 29.8% of urban older men . concurrently , 37.4% of urban older men reported that they were sometimes satisfied with life compared to 30.2% of rural men . twenty - nine percent of urban men indicated that were satisfied with life most times compared to 30.5% of rural men . a cross tabulation between life satisfaction and happiness revealed a significant statistical correlation - 2 ( df = 9 ) = 1334.448 , p < 0.001 . the association was a relatively strong one ( correlation coefficient = 0.663 ) table 6 . seventy - three percent of those older men who were rarely happy were rarely satisfied with life compared to 17.8% who indicated being always happy that were rarely satisfied with life . forty - seven percentages of those who were always satisfied with life were always happy . further investigation revealed that 2.1% of those who were always satisfied with life were rarely happy . life satisfaction by happiness based on table 7 , the model ( eqn . ) is a good fit for the data f ( 19 , 1855 ) = 6.492 , p < 0.00 . continuing , 36.2% of the variance can be explained by age of respondents ; social support ; church attendance ; area of residence ; the number of people in the household and depression . using beta weights , church attendance was the most significant predictor of functional capacity ( =0.245 ; 95% ci : 0.264 , 1.291 ) followed by social support ( =0.129 ; 95% ci : 0.129 , 0.258 ) , area of residence ( =-0.060 ; 95% ci : -0.427 , -0.061 ) and furthermore , urban older men in jamaica had a low functional capacity than rural men ; and that the older men becomes their functional capacity falls - elderly ( ages 64 to 74 years , =-0.051 ; 95%ci : - 0.427 , -0.009 ) ; elderly ( ages 75 years and older , =-0.054 , 95%ci=-0.523 , -0.013 ) . multiple regression of functional status by some explanatory variables , n=1,875 most of the sample was lowly dependent ( 77.1% ) ; 55.4% reported a moderate health status and 63.6% indicated satisfied with life sometimes ( table 2 ) . demographic characteristics of sample when functional capacity was disaggregated into adl and ( i ) adl , the following disparities were observed in the findings . of the sample , however , with regards to ( i ) adl , 1.9% had low , 18.6% moderate and 79.6% high scores . of the sample , 43.1% reported that they were suffering from depression compared to 56.9% who stated no to the question of being depressed in the survey period . on examining depression and age cohort , no significant statistical association was between both variables ( p = 0.102 ) . one half of the sample indicated that they spent ja.$100 ( us $ 1.45 ) monthly for medical expenditure ; 34% of the respondents bought their prescribed medication ; 17.1% reported that they have been hospitalized since their sixth birthday and 65.8% reported that they took no medication . of those who mentioned that they were ill during childhood ( 17.5% , n=350 ) , 34.9% said that the illness was measles or chicken pox , 26.3% mentioned asthma , 10.0% pneumonic fever , 8.9% polio , 6.6% accident , 4.6% jaundice , 1.7% hernia , and 5.1% indicated gastroenteritis . twenty four percent of elderly men indicated that they were rarely happy , 40.5% said sometimes , 31.0% mentioned often and only 4.5% reported always . furthermore , 17.7% of the sample reported that they were seriously ill as children . the findings revealed that no statistical correlation existed between adl and age cohort of the sample ( p=0.205 ) . however , a relationship was found between ( i ) adl and age group of the sample ( 2 ( df = 4 ) = 16.011 ; p=0.003 ) ( table 3 ) . on further examination , it was revealed that as an older male increases in age from 55 - 64 years to 65 - 74 years and 75 + years , his high independence falls and while his moderate dependence increases . of those who were 55 - 64 years , 83.0% of them high independence compared to 78.9% of those 65 - 74 years and 73.9% for those 75 + years ( table 3 ) . adl and ( i ) adl by age group of the sample , 41.8% of older men were health literate or have been advised on medical relates conditions , causes , prognosis and precautions compared to 58.3% who were not aware or have been advice by a health care practitioners ( include a pharmacists , community aide ; nurse , or medical technologists ) . the study revealed that no statistical correlation was found between functional capacity of older men in jamaica and health advice ( or health literacy ) p = 0.845 . however , a weak statistical relationship existed between educational level and health literacy ( 2 ( df = 1 ) = 110.165 , p < 0.001 , correlation coefficient = 0.235 ) ( table 4 ) . health literacy by high level of education attained table 5 revealed that adl for men 55 + years was very high for each activity , with 88.5% for continence being the lowest level of independence . for ( i)adl , 56.7% of sample was still able to perform heavy duty housework , 62.7% were still performing their laundry , 98.1% managed their money and 77.8% were still shopping , and 70.2% prepared their own meals . disaggregating adl and ( i ) adl of sample no statistical correlation was found between health status and age cohort of respondents ( p=0.051 ) , and between life satisfaction and age cohort ( p = 0.430 ) as well as health status and area of residence ( p = 0.190 ) . a significant statistical difference between life satisfaction of urban and rural older men in jamaica ( df = 3 ) = 13.910 , p = 0.003 . however that the correlation was a weak one ( correlation coefficient = 0.083 ) . on further examination , 35.9% of older rural men revealed that they were rarely satisfied with life compared to 29.8% of urban older men . concurrently , 37.4% of urban older men reported that they were sometimes satisfied with life compared to 30.2% of rural men . twenty - nine percent of urban men indicated that were satisfied with life most times compared to 30.5% of rural men . a cross tabulation between life satisfaction and happiness revealed a significant statistical correlation - 2 ( df = 9 ) = 1334.448 , p < 0.001 . the association was a relatively strong one ( correlation coefficient = 0.663 ) table 6 . seventy - three percent of those older men who were rarely happy were rarely satisfied with life compared to 17.8% who indicated being always happy that were rarely satisfied with life . forty - seven percentages of those who were always satisfied with life were always happy . further investigation revealed that 2.1% of those who were always satisfied with life were rarely happy . life satisfaction by happiness based on table 7 , the model ( eqn . ) is a good fit for the data f ( 19 , 1855 ) = 6.492 , p < 0.00 . continuing , 36.2% of the variance can be explained by age of respondents ; social support ; church attendance ; area of residence ; the number of people in the household and depression . using beta weights , church attendance was the most significant predictor of functional capacity ( =0.245 ; 95% ci : 0.264 , 1.291 ) followed by social support ( =0.129 ; 95% ci : 0.129 , 0.258 ) , area of residence ( =-0.060 ; 95% ci : -0.427 , -0.061 ) and furthermore , urban older men in jamaica had a low functional capacity than rural men ; and that the older men becomes their functional capacity falls - elderly ( ages 64 to 74 years , =-0.051 ; 95%ci : - 0.427 , -0.009 ) ; elderly ( ages 75 years and older , =-0.054 , 95%ci=-0.523 , -0.013 ) . multiple regression of functional status by some explanatory variables , n=1,875 most of the sample was lowly dependent ( 77.1% ) ; 55.4% reported a moderate health status and 63.6% indicated satisfied with life sometimes ( table 2 ) . demographic characteristics of sample when functional capacity was disaggregated into adl and ( i ) adl , the following disparities were observed in the findings . of the sample , however , with regards to ( i ) adl , 1.9% had low , 18.6% moderate and 79.6% high scores . of the sample , 43.1% reported that they were suffering from depression compared to 56.9% who stated no to the question of being depressed in the survey period . on examining depression and age cohort , no significant statistical association was between both variables ( p = 0.102 ) . one half of the sample indicated that they spent ja.$100 ( us $ 1.45 ) monthly for medical expenditure ; 34% of the respondents bought their prescribed medication ; 17.1% reported that they have been hospitalized since their sixth birthday and 65.8% reported that they took no medication . of those who mentioned that they were ill during childhood ( 17.5% , n=350 ) , 34.9% said that the illness was measles or chicken pox , 26.3% mentioned asthma , 10.0% pneumonic fever , 8.9% polio , 6.6% accident , 4.6% jaundice , 1.7% hernia , and 5.1% indicated gastroenteritis . twenty four percent of elderly men indicated that they were rarely happy , 40.5% said sometimes , 31.0% mentioned often and only 4.5% reported always . furthermore , 17.7% of the sample reported that they were seriously ill as children . the findings revealed that no statistical correlation existed between adl and age cohort of the sample ( p=0.205 ) . however , a relationship was found between ( i ) adl and age group of the sample ( 2 ( df = 4 ) = 16.011 ; p=0.003 ) ( table 3 ) . on further examination , it was revealed that as an older male increases in age from 55 - 64 years to 65 - 74 years and 75 + years , his high independence falls and while his moderate dependence increases . of those who were 55 - 64 years , 83.0% of them high independence compared to 78.9% of those 65 - 74 years and 73.9% for those 75 + years ( table 3 ) . adl and ( i ) adl by age group of the sample , 41.8% of older men were health literate or have been advised on medical relates conditions , causes , prognosis and precautions compared to 58.3% who were not aware or have been advice by a health care practitioners ( include a pharmacists , community aide ; nurse , or medical technologists ) . the study revealed that no statistical correlation was found between functional capacity of older men in jamaica and health advice ( or health literacy ) p = 0.845 . however , a weak statistical relationship existed between educational level and health literacy ( 2 ( df = 1 ) = 110.165 , p < 0.001 , correlation coefficient = 0.235 ) ( table 4 ) . health literacy by high level of education attained table 5 revealed that adl for men 55 + years was very high for each activity , with 88.5% for continence being the lowest level of independence . for ( i)adl , 56.7% of sample was still able to perform heavy duty housework , 62.7% were still performing their laundry , 98.1% managed their money and 77.8% were still shopping , and 70.2% prepared their own meals . disaggregating adl and ( i ) adl of sample no statistical correlation was found between health status and age cohort of respondents ( p=0.051 ) , and between life satisfaction and age cohort ( p = 0.430 ) as well as health status and area of residence ( p = 0.190 ) . a significant statistical difference between life satisfaction of urban and rural older men in jamaica ( df = 3 ) = 13.910 , p = 0.003 . however that the correlation was a weak one ( correlation coefficient = 0.083 ) . on further examination , 35.9% of older rural men revealed that they were rarely satisfied with life compared to 29.8% of urban older men . concurrently , 37.4% of urban older men reported that they were sometimes satisfied with life compared to 30.2% of rural men . twenty - nine percent of urban men indicated that were satisfied with life most times compared to 30.5% of rural men . a cross tabulation between life satisfaction and happiness revealed a significant statistical correlation - 2 ( df = 9 ) = 1334.448 , p < 0.001 . the association was a relatively strong one ( correlation coefficient = 0.663 ) table 6 . seventy - three percent of those older men who were rarely happy were rarely satisfied with life compared to 17.8% who indicated being always happy that were rarely satisfied with life . forty - seven percentages of those who were always satisfied with life were always happy . further investigation revealed that 2.1% of those who were always satisfied with life were rarely happy . based on table 7 , the model ( eqn . ) is a good fit for the data f ( 19 , 1855 ) = 6.492 , p < 0.00 . continuing , 36.2% of the variance can be explained by age of respondents ; social support ; church attendance ; area of residence ; the number of people in the household and depression . using beta weights , church attendance was the most significant predictor of functional capacity ( =0.245 ; 95% ci : 0.264 , 1.291 ) followed by social support ( =0.129 ; 95% ci : 0.129 , 0.258 ) , area of residence ( =-0.060 ; 95% ci : -0.427 , -0.061 ) and lastly by age of respondents . furthermore , urban older men in jamaica had a low functional capacity than rural men ; and that the older men becomes their functional capacity falls - elderly ( ages 64 to 74 years , =-0.051 ; 95%ci : - 0.427 , -0.009 ) ; elderly ( ages 75 years and older , =-0.054 , 95%ci=-0.523 , -0.013 ) . functional capacity of older men in jamaica was very high as 77 out of every 100 men 55 + years had a high independence ; 22 out of every 100 a moderate independence , and 1 out of every 100 had a low independence . this is in somewhat lower eldemire 's earlier work that showed that 93.5% percentage of elderly jamaicans were actively involved in daily management of the household ; 88.5% were physically functional , and 85.9% were mentally competent . comparatively though , the functional capacity of elder men with that of elder jamaicans showed that physically functionality of the men 55 + years had fallen by 11.5% in 12 years . on average , the physical functional capacity of older men has been declining by 1% each year since 1995 . using self - reported depression as in indicator of mental functioning , the current research revealed that 4 out of every 10 older men were suffering from depression , suggesting that there is also a decline in mental competency of older men based on eldemire 's earlier work on elders in jamaica . furthermore , older men were predominantly satisfied with life some ; having attained at most primary level education and had good health status . thirty - four out of every 100 older men in jamaica was rarely satisfied with life , with there being more unsatisfied older rural men than urban older men . on disaggregating the current study 's findings , using adl , it was revealed that most of men 55 + years were able to bathe , feed , use toilet , and dress themselves with minimal assistance . however , using ( i)adl , which measures activities that people can continue accomplish independently in their residence in the community , it was found that 79.6% needed minimal assistance ( high independence ) compared to 18.6% moderate and 1.9% low independence . there are some similarities and differences between older filipinos [ ] , and older men in jamaica . with respect to transportation 18.5% of filipinos had low independence compared to 1.9% of jamaicans older men ; 17.6% were having low independence with shopping compared to 22.3% of older men in jamaica ; 6.3% of filipinos needed assistance dressing themselves compared to 2.2% of elderly jamaican ; 11.5% of filipinos had difficulties walking in the house compared to 2.3% in this study ; and 8.0% of filipinos had difficulties bathing themselves compared to 2.5% in this research . on the matter of self - reported health status , for the current study no older jamaican men reported poor or very good health status , while 17.5% and 5.1% of filipinos reported poor and very good health status respectively . nineteen percentage of older men in sample indicated excellent health compared to 1.0% of older filipinos . with regards to good health status , 55.4% percent of current sample and this was 31.5% of filipinos ; and for fair health status , 25.6% of the current study and 45.0% of filipinos . like natividad and zimmer , the current study found that as older jamaican ages , their high independence in ( i ) adl falls . however natividad and zimmer found a similar result for adl , but this research found that there is not statistical difference between ageing for men 55 + years and adl . generally , there is a high degree of independence of older men in jamaican and older filipinos . unlike the filipinos study that did not examine life satisfaction , the current study found that only 4 out of every 100 men 55 + years were generally satisfied with life most times ; 64 out of every 100 reported sometimes and 33 out of 100 indicated that they were rarely satisfied with life . in this research , 18 out of every 100 men indicated that their health status has a child was poor . ten percent in both elder men in jamaica and older filipinos ( 10.1% ) had no formal education ; 57.1% of latter group lived in urban zones compared to 49% in the current research . another similarity between both studies is the use of 55 + years to collect the data from , which is used to examine a functional model . for the current work , the model can explain 36.2% of the variability in functional capacity of older men in jamaica . although natividad and zimmer 's work did not provide such information , age and area of residence were found to be common predictors of functional capacity in both studies ; however , in natividad and zimmer , an older filipinos was 0.34 times less likely to reported better ( i)adl than an urban elderly . in this study , this was not the case as it was revealed that urban older men 55 + years were less likely to reported better functional capacity than rural older men . the current work when further than natividad and zimmer 's study , by adding some more variables such as depression , number of people living in household ; social support , and church attendance . depression was found to be inversely associated with functional capacity as well as number of people in household . like natividad and zimmer , this study found that marital status and education were not statistically significant determinants of functional capacity ( i.e. adl or ( i)adl ) . however , the significant statistical correlation between church attendance and functional capacity is embedded in the ability to walk or the use of limb functions[1820 ] . hence , the findings is not supporting any perspective that church attendees were healthier ; but that they were highly probable to higher functional independence than non - attendees , and this also the case for those who attend other social institutions . the researcher needs to make the aforementioned distinction as the current research did not seek to investigate when those who attended church were healthier ; but that whether they were more likely to be functionally independent than non - attendees or for that matter those who attend other social networks . hence , the lowered functional capacity of those who aged explain not only reduced activities outside of the home ; but speaks volume about those who are able to attend outside activities ( including church functions ) that they are of higher independence . depression can be used to measure cognitive functionality , and so the negative correlation between depression and functional capacity concurs with the findings in other study that reported the strong correlation between cognitive functions and functional capacity . the matter of depression affects 4 in every 10 older men in jamaica , and with the inverse association between it and functional capacity , there is expected to be a decline in functional capacity of this cohort . although depression and life satisfaction are weakly correlated in this sample , the reality is it is further depleting the quality of life lived by men 55 + years in jamaica , and so offers some insight into the further decline in functional capacity of this cohort . while depression is permanent and to some extent it is fluid , rate of depression in the current older men in jamaica is too high , and offers another explanation for the high mortality of men in the elderly compared to older women . in a study conducted by yi and vaupel of 8,805 elder ages 80 - 105 years in china , self - reported health status was found to be significantly correlated to functioning and morality of older people , which was also found in earlier studies . in spite of those findings , the current study did not concur with those results as it was revealed herein that childhood health status or current health status was not significantly associated with functional capacity . this research also concurs with yi and vaupel 's work that there was no statistical difference between urban - rural residents in current health status . although no statistical correlated was found between the two aforementioned variables , only a minimal number of elder men in jamaica had high dependence on others ( 2 out of every 100 ) and none indicated poor health status . chevannes 's work begins the explanation of the cultural health care - seeking behavior of males therein a broader context of culturalization of boys . chevannes provided the explanation for this behavior by men , that it is embedded with social learning theory . in which the young imitate the roles of society members through role modeling of what constitute acceptable and good roles which is supported by reinforcement . the gender role of sexes is not limited to jamaica or the caribbean but a study carried out by ali and de muynck of street children in pakistan found a similar gender stereotype in that nation . it was a descriptive cross - sectional study carried out during september and october 2000 , of 40 school - aged street children ( 8 - 14 years ) . the sample was substantially males ( 80% ) , with a mean age of 9 years ( 2 years ) . the methods of data collection were ( i ) semi - structured interviews , and ( ii ) a few focus group discussions . ali and de muynck found that the sampled population would seek medical care based on severity of illnesses and financial situation . the reason being that mild ailment is not severe enough to barr them from physical functioning , which mean that they are okay ; and so some morbidity are not for - hospital , which was so the case in nairobi slums . therefore , like the cases in pakistan and nairobi , the jamaican men are equally not reporting illness and seeking health - care substantially because of their socialization . this is tied to the macho culture with which they are grown as pointed out by chevannes that they should suppress response to pain and similarly displayed in ali and muynck and taff and chepngeno works . the current study revealed that a miniscule percentage of older men in jamaica were satisfied with life most - to - always ; many of them were lowly dependent ; few indicated fair health status and no significant statistical correlation was found between adl and age cohort although one existed between ( i ) adl and age groups . the findings revealed that as men age ( i.e. from 55 years ) ; there is deterioration between ageing and ( i ) adl , suggesting the challenges of ageing and some aspects of functional capacity . concurrently , six factors explain functional capacity of older men in jamaica ( area of residence ; age ; social support ; church attendance ; number of people in household and depression ) . more rural men were rarely satisfied with life ; but more of them had a greater functional capacity than urban men . depression was found to negatively relate to functional capacity , and church attendees had a greater functional status than non - attendees .

context : surgical instruments and materials continue to be retained in the peritoneal cavity despite precautionary measures . even though uncommon it is also under - reported and carries serious medico - legal consequences . gauzes and sponges ( gossypiboma ) are the most commonly retained materials and intra - abdominal retained artery forceps are much rarer but when they do occur lead to chronic abdominal pain and can be a rare cause of intestinal obstruction or strangulation with significant morbidity and mortality.case report : we present a case of intraabdominal retained artery forceps in a 70-years - old lady who underwent laparotomy with splenectomy for a large spleen in a peripheral hospital . upon discharge she continued to complain of intermittent abdominal pain of increasing severity . 12 months later she presented to us with an acute ( surgical ) abdomen requiring another laparotomy . at laparotomy she had strangulated / gangrenous lower jejunual and upper ileal bowel loops , the small bowel mesentery of this area being tightly trapped between the jaws of the retained artery forceps . she had gut resection and enteroanastomosis . unfortunately she died from continuing sepsis on the second post - operative day.conclusion:retained instruments in intra - abdominal surgery can cause serious complication and should be treated surgically . high index of suspicion and appropriate investigations like plain abdominal x - ray , abdominal ultrasound and ct and mri scans should be instituted in patients who develop chronic abdominal symptoms following laparotomy . preventive measures against retained instruments must follow strict laid down protocols for surgical instruments handling in theatre . background : an extensive search of the literature found no studies that have examined functional capacity [ activities of daily living ( adl ) and instrumental activities for daily living ( i ) adl ) ] of jamaican older men as well as factors that determine their functional capacity.aims:the current study examines 1 ) adl , 2 ) ( i ) adl ) , 3 ) self - reported health status , 4 ) functional capacity , and 5 ) factors that determine functional capacity of older men.methods and method : stratified multistage probability sampling technique was used to draw a sample of 2,000 55 + year men . a132-item questionnaire was used to collect the data . descriptive statistics provide background information on the sample , cross tabulations were used to examine non - metric variables and logistic regression provides a model of predictors of functional capacity.result:fifty-five percent of sample indicated good current health status . four percent was mostly satisfied with life ; 21.7% had moderate dependence ; 77.1% had high dependence ( i.e. independence ) ; 1.2% had low dependence ; 21.9% were ages 75 years and older ; 35.6% were ages 65 to 74 years and 42.6% reported ages 55 to 64 years . functional capacity can be determined by church attendance ( =0.245 ; 95% ci : 0.264 , 1.291 ) ; social support ( =0.129 ; 95% ci : 0.129 , 0.258 ) , area of residence ( =-0.060 ; 95% ci : -0.427 , -0.061 ) and lastly by age of respondents.conclusion:ageing in explains deterioration in their ( i ) adl , suggesting the challenges of ageing men 's independence . more rural men were rarely satisfied with life ; but more of them had a greater functional capacity than urban men . depression was found to negatively relate to functional capacity , and church attendees had a greater functional status than non - attendees .

they were randomized to placebo , metformin ( 850 mg twice daily ) , or a lifestyle intervention . the participants mean age was 51 years and mean bmi was 34.0 kg / m , 68% were women , and 45% belonged to u.s . the lifestyle and metformin interventions reduced the incidence of diabetes by 58 and 31% , respectively , versus placebo ( 7 ) . in total , 2,994 participants ( 988 on metformin ) consented to genetic investigation . demographic characteristics of the dpp cohort by treatment arm and genotype at rs11212617 we genotyped rs11212617 on a sequenom iplex platform as previously described ( 8,9 ) . diabetes incidence was determined by semiannual measurements of fasting glucose and an annual oral glucose tolerance test . the principal study outcome was the development of diabetes by american diabetes association criteria , including confirmation . besides diabetes incidence as a categorical outcome , we selected the insulin sensitivity index ( isi ) , fasting glucose , a1c , weight , and oral disposition index ( dio ) as indices of metformin response . we calculated the isi as 22.5/([fasting insulin fasting glucose]/18.01 ) ( or 1/homeostasis model assessment of insulin resistance ) ( 10 ) , and the dio as 1/fasting insulin insulinogenic index ( insulin/glucose over the first 30 min of the oral glucose tolerance test ) ( 11 ) . we tested the additive effect of genotype at rs11212617 on diabetes incidence by cox proportional hazards regression models with genotype and intervention and their interactions as the independent variables predicting time to diabetes over mean 3.2 years follow - up , adjusted for sex , ethnicity , treatment arm , baseline age , and waist circumference . we included all three treatment arms and an interaction test to simultaneously rule out a main effect of this variant on diabetes incidence independent of metformin , or under the action of a lifestyle intervention . we used generalized mixed models to test additive effect of genotype on baseline log - transformed traits and , to model change under metformin action , on the same traits after 1 year of intervention adjusted for the baseline value of the respective trait , age , sex , ethnicity , treatment arm , and waist circumference . post hoc power calculations ( which should be interpreted with caution ) show that the sample size in the dpp metformin arm has > 99% power to detect the change in a1c of 0.61% that was reported in the ukpds ( 5 ) . to control for the potential effect of ethnicity , we performed sensitivity analyses in the largest race / ethnic group ( white participants ) , which is most closely related to the populations examined in the original report and whom we have previously shown to be essentially free of non - european admixture ( 12 ) . they were randomized to placebo , metformin ( 850 mg twice daily ) , or a lifestyle intervention . the participants mean age was 51 years and mean bmi was 34.0 kg / m , 68% were women , and 45% belonged to u.s . the lifestyle and metformin interventions reduced the incidence of diabetes by 58 and 31% , respectively , versus placebo ( 7 ) . in total , 2,994 participants ( 988 on metformin ) consented to genetic investigation . diabetes incidence was determined by semiannual measurements of fasting glucose and an annual oral glucose tolerance test . the principal study outcome was the development of diabetes by american diabetes association criteria , including confirmation . besides diabetes incidence as a categorical outcome , we selected the insulin sensitivity index ( isi ) , fasting glucose , a1c , weight , and oral disposition index ( dio ) as indices of metformin response . we calculated the isi as 22.5/([fasting insulin fasting glucose]/18.01 ) ( or 1/homeostasis model assessment of insulin resistance ) ( 10 ) , and the dio as 1/fasting insulin insulinogenic index ( insulin/glucose over the first 30 min of the oral glucose tolerance test ) ( 11 ) . we tested the additive effect of genotype at rs11212617 on diabetes incidence by cox proportional hazards regression models with genotype and intervention and their interactions as the independent variables predicting time to diabetes over mean 3.2 years follow - up , adjusted for sex , ethnicity , treatment arm , baseline age , and waist circumference . we included all three treatment arms and an interaction test to simultaneously rule out a main effect of this variant on diabetes incidence independent of metformin , or under the action of a lifestyle intervention . we used generalized mixed models to test additive effect of genotype on baseline log - transformed traits and , to model change under metformin action , on the same traits after 1 year of intervention adjusted for the baseline value of the respective trait , age , sex , ethnicity , treatment arm , and waist circumference . post hoc power calculations ( which should be interpreted with caution ) show that the sample size in the dpp metformin arm has > 99% power to detect the change in a1c of 0.61% that was reported in the ukpds ( 5 ) . to control for the potential effect of ethnicity , we performed sensitivity analyses in the largest race / ethnic group ( white participants ) , which is most closely related to the populations examined in the original report and whom we have previously shown to be essentially free of non - european admixture ( 12 ) . the frequency of the c ( metformin - responsive ) allele was 42.4 , 72.4 , 40.1 , 51.6 , and 41.5% in 1,669 white , 609 african american , 497 hispanic , 127 asian / pacific islander , and 82 american indian participants , respectively ( table 1 ) . we found no association of genotype with diabetes incidence in all arms combined , either in unadjusted analyses ( hazard ratio [ hr ] per copy of the c allele 0.98 [ 95% ci 0.881.10 ] , p = 0.76 ) or after adjusting for age , sex , ethnicity , and treatment arm ( hr per copy of the c allele 0.95 [ 0.851.07 ] , p = 0.42 ) ; further adjustment for waist circumference produced indistinguishable results ( table 2 ) . though there was a nominal snp metformin interaction in the fully adjusted model ( p = 0.04 ) , the observed trend was in the opposite direction from the expected prevention effect ; the c allele conferred no detectable advantage on metformin recipients in diabetes prevention but was associated with a nonsignificant trend toward increased risk of diabetes ( hr per copy of the c allele 1.17 [ 0.961.42 ] , p = 0.13 ) . we found no significant associations of genotype with relevant quantitative glycemic traits at baseline ; similarly , there were no significant differences across genotype groups in change in isi , fasting glucose , a1c , or dio after 1 year of metformin ( table 3 ) . the c allele was associated with greater weight gain in the metformin arm . in this arm , there were no statistically significant interactions between the c allele and bmi or waist circumference on diabetes incidence . analyses stratified by ethnic group failed to show any ethnic - specific beneficial effects of the c allele with regard to diabetes incidence on metformin - treated participants ( table 4 ) . diabetes incidence in the dpp by genotype at rs11212617 , treatment arm , and self - reported ethnicity association of rs11212617 with quantitative glycemic traits at 1 year ethnic - specific effects of rs11212617 on diabetes incidence in the dpp in the dpp , the effect of metformin to prevent diabetes or improve relevant glycemic traits was not magnified among carriers of the c allele at rs11212617 in the atm gene . our findings do not support the previously reported association of this allele with improved metformin action on glycemic control . the original association was consistent in three different datasets ( the discovery sample and two follow - up cohorts ) and has been reported recently in other clinical cohorts similarly ascertained ( 13 ) . first , metformin response is defined differently in a prediabetic cohort ( impact on diabetes incidence or quantitative glycemic traits ) than it is in a disease cohort ( ability to reach a1c 7% under treatment ) . second , metformin may be more effective in individuals with a higher a1c at baseline , and thereby the effects of genotype on response might be easier to detect in the disease setting . third , the reported effect might be confined to populations of european descent , e.g. , if rs11212617 tags a low - frequency variant unique to white populations , further diminishing statistical power in the dpp multiethnic cohort . and fourth , the previously reported gwas was based on a retrospective evaluation of clinical records , where potential confounders ( e.g. , if genotype were to influence comorbidities that affect patient adherence , continuity of care , or frequency of a1c measurements ) are harder to control than in a clinical trial . to address potential ethnic differences in the genomic architecture of this region that might explain our negative results , we examined the haplotype structure around this locus in the hapmap european ( ceu ) and west african ( yri ) datasets . the full atm gene and the rs11212617 variant share a segment of tight linkage disequilibrium in both the ceu and yri populations ; there is a recombination hot spot downstream from rs11212617 , beyond which snps display equally low correlations with rs11212617 in ceu and yri , indicating that major differences in linkage disequilibrium patterns would be unlikely to account for potentially discrepant findings in europeans and africans . furthermore , the region distal to this hot spot was well captured by the original gwas array , suggesting that a true signal emerging from this region ( and which might have explained a stronger association in europeans than in africans ) would have also been detected by the original gwas . more generally , a better - powered genome - wide assessment of pharmacogenetic responses in t2d is needed ; whether genetic information will prove useful in diabetes prevention or therapeutics must be tested in prospective clinical trials . pentalogy of cantrell is a rare multiple congenital malformation which occurs worldwide with a reported incidence of 5.5 per million live births.1 the exact cause is not known but is mainly thought to be sporadic , though its being associated with some chromosomal disorders like trisomy 1823 and deletion on locus at xq25 - 26 has been described in some cases.4 it was first described by cantrell et al . , in 19585 with the pentad consisting of a giant omphalocoele and a supra - umbilical anterior abdominal wall midline defect , sternal cleft , ectopia cordis , intracardiac congenital malformations like ventricular septal defect , atrial septal defect and tetralogy of fallot , ventral midline diaphragmatic defect with defect of the diaphragmatic pericardim.5 other associated defects include cranial and facial anomalies , clubfeet , malrotation of the colon , hydrocephalus and anencephaly.67 pentalogy of cantrell often have a poor outcome which is dependent on the severity of the malformations.8 though some cases of pentalogy of cantrell had been reported in nigeria , its true prevalence is not known and none of those reported had been proven to completely fulfill the five main components of the pentad.91011 we , therefore , report a case of pentalogy of cantrell in a 9-month - old boy who completely fulfilled the diagnostic criteria . a 9-month - old boy was first seen at the age of 5 months being delivered with multiple congenital malformations involving the anterior abdominal and chest walls . the child was delivered at term gestation and at home ; the pregnancy was not adversely eventful and she had no antenatal care . however , the child has defect on the anterior abdominal wall with a huge swelling extending from the lower anterior abdomen to the lower anterior chest wall , which was pulsatile and covered by a thin membrane which was initially reddish at birth but later became thickened and darker following daily cleaning and dressing . there was no history of vomiting or constipation , child sucked directly from the breast , though interrupted occasionally to catch his breath , there was no history of difficulty with breathing and no darkening of the lips or mucous membrane . the mother was a 25-year - old stay at home mother with four other children in a non - consanguineous marriage ; she neither smoked cigarette nor drank alcohol and had no adverse medical record . , there was a big pendulous and pulsatile mass extending from slightly above the symphysis pubis to the epigastrum measuring 12 10 cm [ figure 1 ] with an epithelised membranous covering ; it had visible peristaltic waves , it was pundunculated , with cardiac pulsation felt in the epigastric region [ figure 2 ] . bowel and heart sounds were heard over the mass below the level of the diaphragm . he was not tachypneic or tachycardic and had first and second heart sounds with a systolic murmur . his chest x - ray showed a midline heart with inferior displacement [ figure 3 ] , abdominal ultrasound showed the swelling to contain loops of bowl and echocardiography revealed a 6 mm secundum atrial septal defect . the child is currently being followed up awaiting staged repairs of the defects . a bulge in the region of the epigastrium and lower sternum x - ray showing loops of bowl in the swelling with displacement of cardiac shadow pentalogy of cantrell is commoner in boys and less than 60 cases have been documented so far worldwide as at 2007.8 the exact mechanism is not completely understood , however , the proposed pathogenesis involves a defect in embryogenesis between 14 and 18 days after conception due to failure of the in folding of the lateral mesoderm . while most affected children die shortly after birth due to the severity of their defect ( especially the associated intra - cardiac defect and risk of infection in open defects ) , it has been reported in a 32-year - old - man.12 our patient was 9-month - old and remained active despite the associated atrial septal defect and has better prospect with adequately staged surgical intervention . our case had all the major features of the pentad , though with a relatively favourable lesion because the membrane of the giant omphalocoele was completely epithelised [ figure 4 ] and the peduncle covered with skin , which helped in preventing infection ; furthermore , the atrial septal defect was of moderate size and the child had remained stable , though he stands the risk of trauma to the barely covered heart , but with proper counselling the child has survived up till this age . the mass covered by thickened , hyperpigmented keratinised membrane pentalogy of cantrell can be diagnosed prenatally but that has not been the case in those reported from nigeria so far because lack of accessibility to a proper antenatal healthcare and for the fact that most of those diagnostic tools are not readily accessible to these low income parents . patients with complete expression of pentalogy of cantrell are rare and few may survive to older age depending on the severity of the associated lesions , more especially the intracardiac defect . furthermore , we advised that patients with giant omphalocoele should be thoroughly evaluated , because they may have an incomplete expression of the syndrome .

objectivethe c allele at the rs11212617 polymorphism in the ataxia - telangiectasia mutated ( atm ) gene has been associated with greater clinical response to metformin in people with type 2 diabetes . we tested whether this variant modified the effect of metformin in the diabetes prevention program ( dpp ) , in which metformin reduced diabetes incidence by 31% in volunteers with impaired glucose tolerance.research design and methodswe genotyped rs11212617 in 2,994 dpp participants and analyzed its effects on diabetes incidence and related traits.resultscontrary to expectations , c carriers enjoyed no preventive advantage on metformin ; their hazard ratio , compared with a carriers , was 1.17 ( [ 95% ci 0.961.42 ] , p = 0.13 ) under metformin . there were no significant differences by genotype in metformin s effects on insulin sensitivity , fasting glucose , glycated hemoglobin , or disposition index.conclusionsthe reported association of rs11212617 with metformin response was not confirmed for diabetes prevention or for effects on relevant physiologic parameters in the dpp . pentalogy of cantrell is a rare congenital malformation whose exact cause is not completely understood ; it is characterized by defects in the anterior abdominal and thoracic walls consisting of omphalocoele , diaphragmatic defect , ectopia cordis , intracardiac defects and sternal clefts . the complex has variable clinical expression with complete and incomplete expressions reported . we , therefore , report a case of complete manifestation of the pentad in a 9-month - old boy .

pustulotic arthro - osteitis ( pao ) was first described by sonozaki.it is a relatively rare disease a 53-year - old woman was admitted to our department in february 2007 with a pustular rash affecting the palms and soles . inflammation and swelling of the small joints and right - sided low back pain developed during hospitalization . the patient had no history of psoriasis and there was no history or clinical evidence of psoriasis in her family . on physical examination , sharply demarcated erythematosquamous lesions with groups of sterile pustules were seen on the palms and soles [ figures 1 and 2 ] . there was diffuse swelling and erythema over the right sternoclavicular , the right wrist and the left ankle joints . pustular lesions on the right palm pustular lesions on the medial sides of feet laboratory findings showed an erythrocyte sedimentation rate ( esr ) of 120 mm / h ( normal < 20 mm / h ) and c - reactive protein ( crp ) of 6.44 mg / dl ( normal<0.8 mg / dl ) . serum was negative for rheumatoid factor and antinuclear factor , and anti - dsdna , anti - ro , anti - la , and anti - sm antibodies were not present . a technetium-99 m diphosphonate - labeled scintigram of bones revealed abnormal focal increases in tracer uptake in the right sternoclavicular and left ankle joints [ figure 3 ] although x - ray of the wrist and the sternocostoclavicular region revealed soft tissue swelling , there was no ossification of the sternocostoclavicular region . a computed tomography scan showed sclerosis of the sacroiliac joints whole - body bone scintigram skin biopsy from lesions on the left hand showed spongiosis and dyskeratotic cells in the basal layer , with infiltration of polymorphonuclear cells under the orthokeratotic epidermis . a dense dermal neutrophilic infiltrate was also present . on the basis of these clinical and histopathologic features and the scintigram findings we made the diagnosis of pao . after about 1 week of treatment , the swelling and pain in the affected joints had decreased , and the prednisone dose was tapered to 10 mg per day . during that time , new palmoplantar pustulotic lesions appeared . the patient is now healthy after 6 months follow - up and no maintenance treatment is required . palmoplantar pustulosis is a skin disease characterized by recurrent eruptions of sterile pustules , with erythema and exfoliation . the lesions are situated exclusively , and often symmetrically , on the palms and/or soles.[13 ] the age of onset is usually 3060 years , and males and females are affected equally . pao is a rheumatic syndrome of unknown etiology and is characterized by an inflamatory osteitis of the sternoclavicular joint along with palmoplantar pustulosis . pao or sonozaki syndrome occurs in about 10%30% of patients with palmoplantar pustulosis . until a few years ago , this syndrome was considered to be a manifestation of psoriatic arthritis and was then erroneously classified by rheumatologists in the sapho ( synovitis , acne , pustulosis , hyperostosis , osteitis ) syndrome . similar clinical features have been observed in patients with sternocostoclavicular hyperostosis ( scch ) and sapho . scch is a rare condition characterized radiologically by progressive hyperostosis of the medial ends of the clavicles , upper ribs , and sternum , together with soft tissue ossification between the upper ribs and clavicles . clinically , the patients present with painful swelling of the sternum , clavicles , and upper ribs . sapho presents with anterior chest wall involvement , axial skeletal lesions , and osteitis of appendicular bones . pao is now classified as a member of the seronegative spondyloarthritis group of disease ( which includes ankylosing spondylitis , reiter 's syndrome , and psoriatic arthritis ) ; however , it is not associated with hla - b27 . the principal lesion in pao is sternoclavicular involvement , with lesions also occurring in the spinal column and the sacroiliac and peripheral joints . our patient had swelling of the sternoclavicular , right wrist , and left ankle joints , as well as low back pain . although our patient had osteoarthritis affecting the sternoclavicular region , right wrist , and left ankle , as shown by the bone scan , there were no obvious radiological changes . arthritis of the sternoclavicular joint is most frequent in patients with pao , but occurs in less than 15% of patients with ankylosing spondylitis , reiter 's disease , and psoriatic arthropathy.[68 ] in the majority of the cases , the arthritis is of seronegative mono- or oligo - arthritic type , as in our patient who had oligoarticular arthritis . in pao , the most commonly affected peripheral joints are the metacarpophalangeal joints , the proximal interphalangeal joints of fingers , and the elbow and knee joints . other joints such as the hip , the ankle , and the wrist joints have also been involved rarely . our patient had ankle and wrist arthritis , but the knee joint was not involved . pao is characteristically non - erosive and transient and does not induce contracture deformities , and this was true in our patient also . the interval between the onset of skin eruptions to the onset of arthro - osteitis is about 2 years in more than 70% of cases . in our patient , the cause of these abnormalities , like the cause of the underlying disease process , remains unclear . recently cultured propionibacterium acnes from biopsy specimens of patients with this syndrome in approximately 50% . this agrees with the experience of other workers , who have also failed to isolate any specific microorganism from the bones or skin lesions of affected patients . in pao , the arthropathy is not associated with hla - b27 , and this was true in our patient also . to the best of our knowledge , hla - b27 is related to generalized pustular psoriasis , psoriatic arthritis , ankylosing spondylitis , and reiter 's disease . x - ray findings have been consistently reported to be negative , except for soft tissue swellings in acute phases , as in our patient also . the presenting symptoms and findings in our patient , including palmoplantar pustulosis and arthro - osteitis , were compatible with sonozaki syndrome or pao . treatment is usually symptomatic and consists mainly of analgesics and nonsteroidal anti - inflammatory drugs such as phenylbutazone and indomethacin . corticosteroids , colchicine , sulfasalazine , methotrexate , and cyclosporine have been used with variable success . topical steroids , etretinate , and topical puva have been used for the pustular lesions on the palms and soles . if the anterior chest wall symptoms are so severe as to interfere with the patient 's daily activities , surgical resection of the clavicle is recommended . presented here , dapsone treatment was used successfully to treat the cutaneous manifestations and it also maintained the prolonged remission . to the best of our knowledge , this is the first report of pao from turkey . radiological studies must be performed in a patient with palmoplantar pustulosis and swelling of joints to avoid missing the diagnosis . shingles , also called herpes zoster ( hz ) , is a common viral disease . psoriasis is another common , chronic relapsing and remitting inflammatory disease that involves the skin and joints with an overall prevalence of 2% to 3% of the world 's population . koebner phenomenon ( kp ) , also called isomorphic response , is initially referred to the formation of psoriasiform lesions after cutaneous trauma on healthy skin areas of psoriatic patients , and now is extended to the instances that the people who had pre - existing dermatosis develop lesions after trauma or injury . only a few of psoriatic kp following shingles have been reported in literature , and the kp lesions always occur at healing or healed hz eruptions with a latent period of 1 week to 4 months from the occurrence of shingles in this condition . herein , we report a psoriatic patient in whom kp is occurring at the site of developing hz lesions , and to our knowledge , no similar description has been reported before . a 30-year - old man was referred with 3-day history of clustered scaly papules on the right chest wall . the patient had episodes of groups of erythematous papules with shooting pain distributed on his right side of chest alone in the dermatome 9 days ago , which quickly turned into vesicles within 2 days . he was diagnosed as hz and started on treatment of valaciclovir on the 4 day after the appearance of painful papules , resulting in rapid pain relief after being treated for 2 days . on the 6 day of the course of disease , the patient found that the vesicles began subsiding and became scaly papules rapidly in situ . the patient had 7-month history of scalp psoriasis showing good response to topical application of calcipotriol ointment , but he used the medicine irregularly resulting in recurrent lesions sometimes . he never had kp lesions , and any other associations , since the onset of psoriasis . cutaneous examination revealed that scaly erythematous papules and plaques located on the scalp and forehead ( figure 1a ) , and groups of clustered erythematous papules with silver scales in the dermatome distributed on the right side of chest wall where the prior hz lesions occurred ( figure 1b , c ) . no other kinds of lesions were observed elsewhere including limbs , nails , and the trunk except the site where the prior hz eruptions involved . the patient was diagnosed as kp of psoriasis , and was treated with topical application of clobetasol propionate and calcipotriol compounds , which resulted in excellent response after a week of treatment ( figure 1d ) . on 1 year of follow - up , the patient had occasional recurrence of psoriasis when the treatment was stopped ; no new zosteriform eruptions reoccurred . interestingly , the recurrent lesions occurred only on the scalp and the areas of prior shingles each time ( figure 1e , f ) . histology from thoracic lesion showed regular epidermal hyperplasia with test - tube - shaped rete ridges , thinning over some derma papilae , acanthosis , parakeratosis , and lack of granular layer ( figure 2 ) . ( b , c ) groups of clustered papules with silver scales in the dermatome distributed on the right side of chest wall where the previous developing hz involved . ( d ) the scaly papules relived markedly after 1 week of topical application of clobetasol propionate and calcipotriol compounds . ( e , f ) the recurrent psoriatic lesions on the areas of the previous shingle on 7 months of follow - up . histology shows regular epidermal hyperplasia with test - tube - shaped rete ridges , thinning over some derma papilae , acanthosis , parakeratosis , and lack of granular layer . the present patient had groups of painful papules and vesicles distributed in the dermatome on unilateral chest wall ; hz can be diagnosed clinically although without laboratory support . generally , the natural history of hz is that its initial lesions appear as erythematous papules , which turn into vesicle within 12 to 24 hours . the vesicles of hz progress into pustules in about 3 days and form scabs over the next 7 to 10 days . the inflammatory lesions within the involved dermatome after hz have been rarely described , but the lesions are present as flat topped lesions or annular papules , and usually appear within a month with the absence of scales . it was found in the present patient that his blisters became scaly papules on the 6 day during the course ; we considered that the scaly papules formed during developing stage of hz rather than recovering time . based on the psoriasis history and the present clinical features : the developing painful papules and vesicles became scaly papules with underlying bleeding points , which showed excellent response to topical application of clobetasol propionate and calcipotriol compounds , we considered that the present scaly papules are kp of psoriasis subsequent to developing hz rather than to healing hz lesions themselves , although slight scarring may occur in patient with hz at the site the blisters have been . the diseases tending to koebnerization include psoriasis , vitiligo , lichen planus , darier disease , bullous dermatoses , and so on . the provoking factors for kp include trauma , burns , friction , insect bites , surgical incision , as well as dermatoses and therapeutics , such as hz , syphilis , and ultraviolet b treatment , and even the cupping therapy , a kind of traditional chinese medicine . season , disease severity , trauma on skin , scar tissue , and emotional stress can also trigger kp in patient with psoriasis . the duration from injury to occurrence of kp may range from 3 days to years , but in general , it is between 10 and 20 days . only a few cases have described their kp lesions of psoriasis at the site of healed or healing hz as well as varicella involved . in the present patient , kp eruptions occurred at the areas of developing hz lesions with only 6-day interval from the painful chest and 4 days from the appearance of vesicles , which , to our knowledge , has never been reported before . other interesting entity is that , on a year of follow - up , the recurrent psoriatic lesions located only on the scalp and the healed hz areas . as the recurrent thoracic lesions occurred synchronously with the scalp 's each time , we considered them to be relapsing psoriasis rather than recurrently psoriatic kp on healed hz . however , the reason why the recurrent psoriasis was localized to the areas mentioned above was unknown . although kp is a common condition , its pathogenesis is not fully understood yet , but may involve cytokines , stress proteins , adhesion molecules , and autoantigens . it is considered that nerve growth factor , tumor necrosis factor- , substance p as well as interleukin-1 play important roles in the pathomechanism of koebnerization in psoriasis . the occurrence of psoriatic koebnerization at the site of shingles and varicella was considered to be mediated by neuropeptides such as substance p. substance p , an endogenous neuropeptide being potentiated by viral infection and acting as a chemomediator of nociceptive impulse from periphery to central nervous system , plays an important role in the hz - associated pain . viral infection can potentiate the effect of substance p by decreasing the degradation of its breakdown enzyme . we speculate that substance p may play an important role in the present kp , although the exact mechanisms remain unknown . interestingly , we soon met another typical shingles patient with prior severe psoriatic lesions over his whole body , but neither his prior shingles involved areas nor other areas occurred kp during 6 months of follow - up . it suggests that kp occurs at the site of hz lesions in not all the psoriatic patients with shingles . as a matter of fact , although both psoriasis and shingles are common diseases , few of psoriatic kp subsequent to hz have been reported . koebner lesions should be treated in the same way as the associated dermatosis , as our present patient did . the present patient was notable because the psoriatic kp lesions occurred at the site of developing hz , and the recurrent psoriasis localized only on the primarily areas and healed shingles areas . both psoriasis and shingles are common cutaneous diseases . about 25% of the patients with psoriasis not only healing and healed shingles can cause kp in psoriasis , but also developing shingles can cause such a condition at the site of hz lesions . the authors thank professor rashmi sarkar , md , department of dermatology , maulana azad medical college and lnjp hospital , new delhi , india , for her assistance in revising the manuscript ; and also thank the patient participating in the present study .

pustulotic arthro - osteitis , first described by sonozaki , is a relatively rare disorder . its prevalence is however probably underestimated in dermatological literature . early recognition of the signs can prevent misdiagnosis . we describe a turkish patient who presented with palmoplantar pustulosis and involvement of the sternoclavicular joint and peripheral oligoarthritis . abstractboth shingles and psoriasis are common cutaneous diseases . about 25% of the psoriatic patients develop koebner phenomenon ( kp ) after various injuries , and in rare instance , kp may occur at the site of healed or healing shingles.we report a 30-year - old man with 7-month history of scalp psoriasis who developed kp at the areas of developing shingles . cutaneous examination revealed scaly erythematous papules and plaques located on the scalp and forehead , and groups of clustered erythematous papules with silver scales in the dermatome distributed on the right side of chest wall the prior herpes zoster lesions involved . after removal of the scales on the papules , underlying bleeding points were present.the lesions on chest had good response to anti - psoriatic therapies , as the lesions on scalp did . after a year of follow - up , recurrent psoriasis occurred , but the lesions were located only on the scalp , and the areas of prior occurrence of shingles , because of which we considered diagnosis of recurrent psoriasis rather than relapsing kp for the chest lesions.not only the healing and healed shingles can trigger kp in psoriasis , but also the developing shingles can cause psoriatic kp at the site of herpes zoster lesions .

subtilosin is a ribosomally - synthesized cyclopeptide produced by bacillus subtilis atcc 6633 and a recently identified natural isolate of bacillus amyloliquefaciens [ 1 , 2 ] . this protein has a unique structure among bacteriocins , and possesses antimicrobial activity against a variety of pathogenic organisms , including gardnerella vaginalis , listeria monocytogenes , and streptococcus agalactiae ( group b streptococcus ) . the ability to inhibit the growth of g. vaginalis is of particular importance as it is one of the primary causative agents for bacterial vaginosis ( bv ) , a common condition found in up to 30% of women in north america . bv is characterized by the replacement of normal vaginal lactobacilli with anaerobic bacteria ( e.g. , prevotella , bacteroides , and mobiluncus ) and mycoplasmas , as well as a several log increase in overall bacterial growth [ 57 ] . bv is asymptomatic in approximately one half of all cases , but is associated with a wide variety of symptoms and problems ranging from complications with pregnancies ( i.e. , preterm births , low fetal birth weight ) to the development of pelvic inflammatory disorder [ 711 ] . recent studies have estimated that nearly one in three women in the united states ( 29.2% ) suffer from bv , with varying prevalence according to ethnicity and education levels . one of the most troubling aspects of bv infection is the association with an increased risk of several sexually transmitted diseases ( stds ) , including chlamydia , herpes , gonorrhea , trichomoniasis , and hiv / aids [ 1216 ] . the treatments recommended by the centers for disease control ( cdc ) are clindamycin and metronidazole administered either orally or intravaginally . following these guidelines successfully treats 60% of bv cases , but 20% of these cases return with highly developed antibiotic resistances [ 1820 ] . in such cases , it would be extremely desirable to have an alternative form of treatment that could fully eradicate the infection . bacteriocins are typically divided into two major , yet diverse , classes : class i , or lantibiotics , contain unusual amino acids and are subject to extensive posttranslational modifications ; and class ii , the heat stable nonlantibiotics . subtilosin has a unique posttranslational structure that is unmatched among bacteriocins , which has led to speculation that it may belong in a distinct and separate class [ 2 , 3 ] . bacteriocins have been widely considered for use in medicinal and pharmaceutical applications , particularly for their bactericidal activity against multidrug resistant organisms . they are especially appealing alternatives since their cost of production is so comparatively low to conventional pharmaceutical treatments . much attention has recently been focused on the spermicidal activity of bacteriocins due to their targeted antibacterial activity and lack of effect on human tissues . nisin a , a well - studied class i bacteriocin produced by lactococcus lactis subsp . lactis , has human spermicidal activity [ 24 , 25 ] , and subtilosin has spermicidal activity against boar , bovine , horse , and rat spermatozoa . the results of these previous studies suggest that subtilosin may also have spermicidal activity against human spermatozoa , prompting our investigation . since subtilosin has proven antimicrobial activity against the pathogen largely responsible for bv , its toxicity to human tissues was assayed to determine its potential as a safe alternative remedy . for the human in vitro study , the epivaginal ectocervical tissue model ( mattek corporation , ashland , mass , usa ) was employed to examine the relationship between prolonged exposure to subtilosin and cell viability . spermicidal evaluations were conducted to investigate the ability of the peptide to restrict or eliminate sperm mobility , leading to its classification as a spermicidal agent . subtilosin was prepared as previously described . to prepare a cell - free supernatant ( cfs ) , cells were removed by centrifugation ( hermle z400k ; labnet , woodbridge , nj , usa ) for 25 minutes at 4500 g and 4c . the supernatant was filter sterilized using 0.45 m filters ( fisher , pittsburgh , pa , usa ) . the protein of interest was precipitated from the supernatant by adding 30% ammonium sulfate ( w / v ) while stirring overnight at 4c and was resuspended in 20 ml of double distilled h2o . the column chromatography method described by sutyak et al . was used to purify subtilosin from the cfs , producing a near - pure isolate in the 90% methanol eluate . the antimicrobial activity of all samples was confirmed by the well - diffusion assay according to the protocol of cintas et al . with additional modifications . the active fraction was concentrated to dryness using a savant sc110 speed vac and uvs400 universal vacuum system ( savant instruments , farmingdale , ny , usa ) , then resuspended in 1.5 ml ddh2o . the concentration of subtilosin in the column - purified fraction was determined using the micro bca protein assay kit according to the manufacturer 's protocol ( pierce , rockford , ill , usa ) . in brief , the assay measures the reduction of cu to cu by colorimetric detection of cu by bicinchoninic acid . bovine serum albumin ( bsa ) was used to develop a standard curve with concentrations ranging from 0.5 to 20 g / ml ; the concentration of subtilosin was calculated using the r value from the trendline of the standard curve graph . the concentration of subtilosin in the cfs was not measurable with the micro bca protein assay due to the high level of background proteins in the solvent ( mrs medium ) . as an alternative , the protein concentration was calculated by comparing the antimicrobial activity of known concentrations of column - purified protein to equal volumes of cfs . five two - fold dilutions were made from the stock samples of both the cfs and the column - purified fraction . well diffusion assays were performed using 50 l of each dilution against micrococcus luteus atcc 10420 , which is commonly used as a reference microorganism for the determination of a bacteriocin 's biological activity . as reported previously , the concentration of lactic acid in the cfs was measured to assess its potential effects on antimicrobial activity and cell viability . the quantity of each form of the acid in the sample was measured using a commercially available d - lactic acid / l - lactic acid kit ( roche boehringer , mannheim , germany ) , according to the manufacturer 's instructions . the epivaginal ( vec-100 ) ectocervical tissue model ( mattek corporation , ashland , mass , usa ) was used and maintained as fully described by dover et al . . the tissues were exposed to 83 l of subtilosin cfs ( 136 g / ml ) for 4 , 24 , and 48 hours . for exposure times over 24 hours , the tissues were aerated by placing them on two metal washers ( mattek corporation , ashland , mass , usa ) and fed with 5 ml of the assay medium . double - distilled water ( ddh2o ) was used as a negative control , and was applied to cells after 6 , 24 , and 48 hours . a spermicidal product containing 4% nonoxynol-9 ( ortho options conceptrol vaginal contraception gel , advanced care products , skillman , nj , usa ) was used as a positive control based on its documented cytotoxic properties [ 30 , 31 ] . a cream ( monistat-3 , ortho mcneil pharmaceutical , inc . , raritan , nj , usa ) containing 4% of the nontoxic , bv - active compound miconazole nitrate [ 30 , 31 ] , was used as a negative control . following the designated exposure times , the mtt ( 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ) assay was used to determine overall cell viability . the data were used to approximate an effective time ( et ) that would reduce cell viability to 50% ( et-50 ) . the mtt assay was carried out according to the protocol outlined by dover et al . . briefly , the viability of ectocervical cells after exposure to subtilosin was measured as a direct proportion of the breakdown of the yellow compound tetrazolium to the purple compound formazan , since only living cells can cause this reaction to occur . tissues were exposed to subtilosin and the two controls for several designated time points ; at the conclusion of each , the liquid in the plate wells was combined with the liquid from the tissue inserts . this mixture was then assayed spectrophotometrically using a 96 well - plate reader ( mrx revelation , dynex technologies , va , usa ) to determine the level of tetrazolium degradation . the viability ( % ) of the treated tissue inserts was calculated according to an equation provided by the manufacturer : % viability = od570 ( treated tissue)/od570 ( negative control tissue ) . the exposure time that reduced tissue viability by 50% ( et-50 ) was calculated according to [ v = a + b * log(t ) ] described by ayehunie et al . , where v = % viability , t = time in minutes , and a and b are constants representing the viability data from the time points preceding and following 50% viability . on the whole , there is a direct relationship between the length of the et-50 and the toxicity of the tested application ( i.e. , a shorter et-50 corresponds to a more harmful compound ) . the cfs gathered from a b. amyloliquefaciens culture was used to test the effect of subtilosin exposure on the motility of human spermatozoa . initially , the cfs was diluted with normal saline ( 0.9% ) so that 200 l of the final material was equivalent to 50 l , 100 l , or 200 l of undiluted cfs . each sample was collected by self - masturbation in a polypropylene specimen container ( fisher ) prior to transport to the laboratory . within 1 hour of collection , total sperm count was calculated using bright field light microscopy ( olympus bx50 ; 400x ) after dilution ( 1:50 ) of the semen in normal saline . the initial percentage of motile sperm was calculated prior to testing with a neubauer hemacytometer . the determination of motile sperm % was made using randomly selected field views ( 400x ) from a count of between 104201 cells . any visibly moving spermatozoa ( directional or stationary ) were counted as motile cells . the percentage of forward progressing spermatozoa was subjectively determined based on the assumption that 70% of the sperm in a normal sample would behave in such a manner . a modified sander - cramer test was used to determine the effect of column - purified subtilosin on human spermatozoa motility . this measured the effect of subtilosin after 30-second exposure times of 5 volumes ( 200 l ) of the solution at each dilution ( 25% and 50% in normal saline , and 100% ) with one volume ( 40 l ) of whole semen . the motilities of cells from random high - magnification fields ( 400x ) of the sample were determined in duplicate as described above . statmost32 ( version 4.1 ) statistical software ( datamost corporation , sandy , utah , usa ) was used to calculate all statistical parameters . any differences between treatment groups were assessed by the newman - keuls multiple range test . the concentration of subtilosin in the column - purified sample was estimated at 135.7 g / ml . the cfs and column - purified sample produced identical zones of inhibition at each dilution ( data not shown ) ; therefore , the concentrations of protein in both solutions were assumed to be equivalent . while it is improbable that a 100% yield would be attained from column chromatography , previous work has shown that protein concentrations can be precisely calculated based on the comparisons we conducted . due to the difficulty in measuring the cfs protein concentration via other assays , the chosen method was deemed the most accurate and reproducible . after 48 hours of exposure to subtilosin , the epivaginal ectocervical tissues retained a high level of viability compared to the positive control , nonoxynol-9 , and the negative control , miconazole nitrate ( table 1 ) . due to the lack of toxicity of the antimicrobial , the et-50 value for subtilosin could not be established since the total cell viability did not drop below 50% at any of the given time points . however , a projection of the et-50 value is possible by an extrapolation of the data . data presented in table 1 can be fit to a curve described by ln(v ) = a + bt , where a = 4.605995356 and b = 0.00014151 ( coefficient of determination , or r , = 0.9998 ) , from which the et-50 is estimated at 70 hours . subtilosin reduces human sperm motility in a dose - dependent manner ( figure 1 ) . the motility of the treated spermatozoa ranged from 0 to 88% of control motility levels over the four - fold range of subtilosin concentrations . all of the subtilosin concentrations tested reduced motility compared to the control samples . the differences in the proportion of motile spermatozoa in all samples ( 28.3 , 56.7 , and 113.3 g / ml protein equivalents ) were found to be significant ( p < .05 ) according to the newman - keuls multiple range test . tablecurve 2d ( ver 5.0 ) curve - fitting software ( spss scientific software , chicago , ill , usa ) was used to fit the data to a dose - response curve described by ln ( % motility ) = a + b [ subtilosin a]3 , where a = 4.20781 ; b = 2.5814e 06 ; and [ subtilosin a ] is expressed as g / ml protein equivalents . the ic50 value , or the amount of subtilosin required to reduce the motility of spermatozoa in whole semen by 50% , was calculated to be 64.5 g / ml . similar to motility , forward progression of spermatozoa is reduced in a dose - dependent fashion by subtilosin . in control samples , 70% of sperm exhibited forward progression ; in the presence of 50 l subtilosin this decreased to 5070% , while 100 l caused a decline to only 10% forward progression . all forward progression was eliminated after treatment with 200 l subtilosin , with most sperm tails becoming coiled . the b. amyloliquefaciens - produced bacteriocin subtilosin has proven antimicrobial activity against the vaginal pathogen g. vaginalis , but was not harmful to the normal and healthy lactobacillus vaginal microbiota . data from human vaginal cell viability assays convincingly demonstrated the safety of subtilosin for human applications in comparison to other accepted and available products , indicating it could be safely incorporated into personal care applications aimed at the treatment of bacterial vaginosis . prior research in our laboratory that involved similar studies with the epivaginal model was also carried out in conjunction with in vivo testing of the rabbit vaginal irritation ( rvi ) system , which doubly confirmed the safety of another antimicrobial peptide , lactocin 160 . therefore , we are confident that using the epivaginal model instead of animal testing to demonstrate the safety of subtilosin has provided reliable and valid results . subtilosin was also found to significantly reduce the motility of human spermatozoa in a concentration - dependent manner for all concentrations tested . the effect of subtilosin on the forward progression of spermatozoa was also observed to be a dose - dependent interaction . serial dilutions showed a steady decline in forward progression , with all progression halted at the highest concentration tested . it was also noted that at the highest concentration , the tails of the sperm cells were curved or coiled , indicating the cells were damaged beyond a simple restriction of movement . coiling of the cells is considered to be a sperm abnormality , and may indicate damage to the plasma membrane . tail coiling has been observed after in vitro exposure of monkey spermatozoa to methyl mercury . it is worth noting that nisin , a bacteriocin given generally recognized as safe ( gras ) status by the food and drug administration ( fda ) , was also shown to have impressive spermicidal activity . however , the allure of these results is diminished due to the fact that it also has potent antimicrobial activity against healthy vaginal microbiota ( chikindas et al . , unpublished data ) , a strong detraction from its commercial applicability . therefore , subtilosin 's spermicidal activity , combined with its overall safety to both human tissues and healthy human microbiota , make it a highly recommendable compound for inclusion in topical bv treatments and human contraceptive products . to facilitate the process of its incorporation into contraceptive treatments , additional analysis will be done to determine the reversibility of damage done to the spermatozoa , as well as a time course assay to further elucidate the exact changes effected by subtilosin . lymphangioma is a developmental malformation of lymphatic tissue , characterized by submucosal tumors covered with normal mucosa.1 these benign tumors are commonly found in children , and although they may develop anywhere , the tumors are usually found in the head , neck , and axillary regions , and rarely in the gastrointestinal tract.2 most reported cases of lymphangioma of the colon are solitary ; few cases involve multiple lymphangioma , also known as colonic lymphangiomatosis.1 colonic lymphangiomas are typically asymptomatic , are discovered only incidentally , and usually do not require treatment.3,4,5 however , in some cases , they may cause abdominal pain , bleeding , intussusception , or protein - losing enteropathy , requiring surgical or endoscopic resection.1,6 a 38-year - old man visited our comprehensive medical center for a general check - up . he had no previous medical problems other than diabetes mellitus and essential hypertension , which were diagnosed 1 year prior at another hospital . laboratory test results , including the levels of tumor markers carcinoembryonic antigen and carbohydrate antigen , were all within normal ranges , except the levels of triglycerides at 206 mg / dl ( normal , 10 to 180 ) and hemoglobin a1c at 9.3% ( normal , 4.0 to 6.0 ) . esophagogastroduodenoscopy showed hyperemic mucosal change in the antrum and body of the stomach , with multiple shallow erosions in the se - cond portion of the duodenum . an initial colonoscopy revealed multiple , variably sized cystic mass lesions in the mid - portion of the ascending colon up to the proximal portion of the transverse colon ( fig . computed tomography ( ct ) revealed no abnormalities other than a fatty liver ( fig . a solution composed of hypertonic saline , epinephrine , and methylene blue was injected around some of the cystic masses and biopsy specimens were taken with snare electrocautery ( fig . incision on one of the masses using a dual knife caused clear yellow fluid to flow out from the mass , after which the mass began to shrink . the biopsied tissue revealed normal colonic mucosa and markedly dilated lymphatic vessels in the submucosa . immunohistochemical staining for cd34 and d2 - 40 showed a positive reaction along the lining cells of the dilated lymphatic channels ( fig . cd34 is an endothelial cell marker in different vascular beds in human tissues,7 and the monoclonal antibody d2 - 40 is a marker of lymphatic endothelium.8 d2 - 40 does not stain vascular endothelium . eight months later , the patient underwent a third colonoscopic examination that showed normal colonic mucosa without the protruded lymphangiomatosis previously been observed in the ascending and transverse colon . no scarring or trace of the former lesions lymphangioma is regarded as a developmental malformation of lymphatic tissue ; obstruction or agenesis of lymphatic tissue causes abnormal dilatation and proliferation of the lymphatic channel , leading to the formation of a cystic mass.3,4 secondary lymphangioma formation can be caused by abdominal trauma , lymphatic obstruction , inflammatory processes , cancer , surgery , or radiation . in the present case , colonic lymphangiomatosis was discovered in adulthood , suggesting that the lesion developed secondary to a local disturbance of the lymphatic circulation . however , the patient had no specific history related to colonic lymphangiomatosis , and abdominal ct showed no comorbid disease ; thus , it would be more rational to conclude that lymphangioma developed primarily in the fetal period in this case , and was silent until detection on medical examination . lymphangiomas rarely occur in the abdomen , and the mesentery , omentum , mesocolon , and retroperitoneum are the most common sites.9 the incidence of lymphangioma in the intestinal wall is very low , and few cases of multiple colonic lymphangioma have been reported.1 in the present case , multiple lymphangiomas of the colon , known as colonic lymphangiomatosis , were observed , and had resolved by the time of the third colonoscopic examination . it is considered benign ; no cases of malignant transformation have been reported.1 trials have been conducted to explain how lymphangiomas may be resolved . several investigators have suggested that increased pressure in the lymphatic system may overcome incomplete obstructions . others have hypothesized that resolution may occur through the establishment of alternative routes of lymphatic drainage.10 the latter theory seems to fit well with the present case . it is possible that in the course of making incisions and taking excisional biopsies of the colonic lymphangiomatosis , conduits that drain lymphatic outflow toward the colonic lumen were formed , leading to the complete resolution of the lymphangiomatosis . there is no standard approach to the treatment of intra - abdominal or intraluminal lymphangiomas . historically , both were treated by surgical resection due to uncertainty regarding the nature of the lesion . however , since lymphangioma is a benign tumor , and most intra - abdominal or intestinal lymphangiomas are asymptomatic , they usually do not require treatment.3,4,5 kochman et al.5 suggested that asymptomatic lymphangiomas should be observed rather than treated . large lymphangiomas may produce bowel obstruction , intussusception , bleeding , anemia , and protein - losing enteropathy , in addition to symptoms such as abdominal pain , vomiting , and diarrhea.6 complete surgical excision is considered the treatment of choice for these large , symptomatic lymphangiomas.1,6 for pedunculated tumors < 2 cm , recent reports indicate a trend towards the use of endoscopic polypectomy.11 in the present case , no treatment other than several incisions and excisional biopsies was attempted because the observed tumors were all small , sessile , and did not exhibit symptoms or cause complications . in a follow - up study after the incisions and excisional biopsies , we found that such tumors were completely resolved . our assumption is that conduits were made at some sites of incisions or excisions , and lymphatic drainage through those conduits from the lymphatic channel to the colonic lumen brought about the resolution of the colonic lymphangiomatosis . during 8 months of surveillance after the procedure , no complications such as bleeding or protein - losing enteropathy were noticed . this is a case report of a patient in whom dozens of lymphangiomas were found in the ascending colon up to the transverse colon , which was observed and confirmed through colonoscopy and biopsy . the colonic lymphangiomatosis was observed to be completely resolved in follow - up studies , without any treatment .

bacterial vaginosis ( bv ) , a condition affecting millions of women each year , is primarily caused by the gram - variable organism gardnerella vaginalis . a number of organisms associated with bv cases have been reported to develop multidrug resistance , leading to the need for alternative therapies . previously , we reported the antimicrobial peptide subtilosin has proven antimicrobial activity against g. vaginalis , but not against the tested healthy vaginal microbiota of lactobacilli . after conducting tissue sensitivity assays using an ectocervical tissue model , we determined that human cells remained viable after prolonged exposures to partially - purified subtilosin , indicating the compound is safe for human use . subtilosin was shown to eliminate the motility and forward progression of human spermatozoa in a dose - dependent manner , and can therefore be considered a general spermicidal agent . these results suggest subtilosin would be a valuable component in topical personal care products aimed at contraception and bv prophylaxis and treatment . lymphangioma is an uncommon malformation of the lymphatic system that involves a benign proliferation of the lymphatics , with no established treatment method . multiple colonic lymphangioma , or colonic lymphangiomatosis , is an extremely rare condition . we report a case of colonic lymphangiomatosis that was detected during a colonoscopic examination conducted as part of a general health check - up . the lesion completely resolved after excisional biopsy .

drawings were collected and analysed as evidence for specific kinds of mental activity in the context of early child psychology , beginning in the 1870s . researchers distinguished children s image production from subjective artistic expression , establishing drawing as a material trace of universal patterns of cognitive development . of course , scribbles , doodles and sketches populate the margins of countless documents belonging to everyone from eighteenth - century schoolboys to us presidents ; in examining scientific uses of drawing , it becomes particularly important to underscore how the simple act of putting pen to paper is historically circumscribed . both in science and in art , changing norms of observation and representation have influenced the way that people draw . in early nineteenth - century europe and america , representational drawing ability belonged to the set of socially valued skills cultivated in elite schools . by the 1820s , a market for drawing instruction developed among the upwardly mobile middle classes , reflected in an explosion of popular how - to books such as samuel prout s easy lessons in landscape drawing ( figure 4 ) . drawing instruction was soon incorporated into the curricula of state - sponsored educational institutions , both for its salubrious influence on character and for its potential industrial applications . source : r. ackermann , easy lessons in landscape drawing ( london : r. ackerman , 1820 ) . source : r. ackermann , easy lessons in landscape drawing ( london : r. ackerman , 1820 ) . as a component of primary education regardless of the strategies that instructors adopted , their efforts established representational drawing as an increasingly standardised visual language . in this vein , david maclagan points out that drawings are linked in different ways to conventional communicative modes , reflecting the established pictorial codes of a particular time and place . however , maclagan also notes that the unconscious reproduction of these codes was understood , starting in the late nineteenth century , as a subliminal kind of pictorial lingua franca revealing the underlying essence of the human mind . indeed , it was precisely in this context a society where most primary school students learned to evaluate the correctness of outline , proportion and shading that drawings could become a taken - for - granted indicator of cognitive development and mental soundness . pioneers in the emerging field of child study turned the pedagogical goals of drawing instructors inside out by asking how the rules of visual language were acquired and what mental architectures they revealed . they documented and classified the process of learning to draw as a proxy for learning to think , based on the proposition that images provided unique access to the developing mind . g. stanley hall was , in large part , responsible for bringing quantitative , developmentally oriented child study to the united states from germany , where the growth of state - sponsored education had produced a vigorous field of pedagogical research by the 1870s . in 1880 , hall asked hundreds of boston school children to produce drawings of everyday objects as part of his inventory of the child mind. the inventory was , quite literally , a laundry - list of things that children knew : animals , places and household objects . the list was generated through one - on - one interviews conducted by elementary school teachers using standardised response forms . most of the prompts were verbal , but hall expressed reservations about this methodology , acknowledging that children s vocabulary did not necessarily correspond with their functional understanding . he thus included drawing tasks in the inventory , claiming that [ d]rawings reveal the child s psychic life in an extraordinary way and show its motor development [ and ] the directions of its interests ... . hall s main interest lay in amassing observations on a scale large enough to reveal generic patterns of the child mind. although hall soon moved on from child study to other endeavours in the emerging constellation of the social sciences , his followers attempted to systematise his methods and build theories from the data they gathered . pedagogical seminary , the journal that hall founded in 1891 , regularly published analyses of children s drawings , written by a growing class of psychologists and educators who had begun to specialise in this area . among the more prolific was herman t. lukens , who served as an honorary fellow under hall at clark university from 1894 to 1895 , and returned as a docent in pedagogy from 1899 to 1900 . lukens collected 3400 drawings from children between two and sixteen years of age , publishing his findings in 1896 . such vast accumulations had become a trend in child study ; some projects netted many thousands of drawings , and requested still more samples from the journal s readers , with the promise that treasured artworks would be duplicated and returned . unlike other traces of the child mind that had to be abstracted from observed behaviour into tables and charts , drawings were printed directly in professional journals utilising a visual rhetoric of immediacy . lukens presented series of drawings done by the same children over the course of many years to illustrate their linear development ; the gradual complete clarification of the mental image into a picture on the page . figure 5:drawing by lillian l. at four years , three months old . source : lukens , a study of children s drawings in the early years , pedagogical seminary 4 , 1 ( 1896 ) 102 . source : lukens , a study of children s drawings in the early years , pedagogical seminary 4 , 1 ( 1896 ) 102 . because the children in lukens s study were so young , he saw their drawings as an unmediated glimpse into their mental architecture , a subjective rather than an objective picture of the world as perceived and understood . a certain level of expertise , however , was required to interpret these often jumbled glimpses . the tangle of lines over the legs is not in any case to be interpreted as shading , he asserted of the child s drawing in figure 5 . such an interpretation would violate the accepted developmental chronology , attributing faculties to the child that experts believed that he or she could not possess . his text framed the drawings with a narrative that included statements of his subjects about their drawing processes and their intentions : the child drew one perpendicular line which she called a leg , then a parallel line which she called the other leg. he also provided reassurances about his control over the experimental setting . although the collection of drawings did not take place in a psychological laboratory , and parents or teachers were often present , lukens inserted narrative asides such as , her mother meanwhile looked on in silence , giving no suggestions , asserting the impartiality of the arrangement . when an adult intervened , lukens was careful to justify the action and minimise its influence on the results . barbara wittmann has emphasised the drastic stripping away of context in early italian and german studies of child art ; later researchers , such as lukens , recognised the need for some context , but maintained tight control over what information framed the material evidence . although lukens spoke of children s drawings as direct projections from their minds , motor development had an undeniable impact on the images . lukens incorporated the relative manual dexterity of children at different ages into his assessment . this opened up the possibility that very young children were , in fact , visualising objects the drawings of lillian l. at the age of two show the utter lack of any apparent connection between a mental picture in consciousness and the movements made by the hand and fingers in attempting to draw it. once lillian l.s drawing ability had improved to the point where she could capture contours , however , lukens readily asserted a correspondence between the acuity of her mental imagery and the accuracy of her drawing : it probably corresponds to a clarification of the mental image in the child s mind by a gradually greater discrimination on her part between the motor elements and the strictly visual elements ... . this clarification of the image by eliminating noise produced drawings of true scientific value . thus , although young children were considered ideal subjects for research into the primitive mind , in practice , they were not so useful during their most primitive stages . the theories of child study experts presupposed a true hand that could accurately relay the mind s messages . they focused on subjects within a particular developmental window who made optimal self - registering instruments . like hall , lukens used children s drawings to argue for a recapitulation theory of human mental development : both saw striking analogies in the pictorial evolution of man between the child and the race. influenced by widespread scientific enthusiasm for ernst haeckel s biogenetic law , they equated child art with the simplistic images produced by primitive cultures , which progressed to the mastery of realism and linear perspective indicative of modern civilisation . lukens , however , sought to nuance this conventional and generic classification of children s drawings . in his work , we see a turn away from hall s statistical abstractions and towards narrative interpretation . although he would not go as far as freud or later promoters of art therapy , lukens swerved into a grey area between the survey style , which considered children s affective states only in so far as they interfered with the registration of accurate responses , and the analytic style which saw drawings as manifestations of a non - verbalised subjectivity . the little child draws from his own consciousness and thus puts his own consciousness into the picture , lukens claimed , meaning that the drawing could not be a pure scientific object in the way that some recapitulationists had supposed . although he considered child art analogous to the pictographic stage in the race development [ that ] has preceded and led up to the alphabetic stage , it also usefully encoded the particulars of a child s experiences , likes and dislikes . it was , paradoxically , a more objective modality than speech for getting at the unique thinking of each child , since words are often repeated meaninglessly , while drawings are pure form synonyms for thoughts. again , lukens asserted that the mind hand relay of the ideal drawing child was free from interference by contextual factors beyond those that he identified as relevant to the investigation of children s interiority . while child study gave way to laboratory - centred developmental psychology in the early twentieth century , the use of drawings to assess children s cognitive ability was formalised in much the way that hall had envisioned . within the first decade after 1900 , drawing tasks as a measure of development had become standard , with systems proposed by frederic l. burke , james mark baldwin , james sully , william stern and many others . in 1926 , draw - a - man test , establishing a widely adopted rubric for correlating children s drawings with their intelligence as measured by the stanford - binet intelligence scale . also in the 1920s , jean piaget developed his influential four - stage theory of artistic development in children , which posited a progression from visual realism. recapitulation fell away as an explicit foundation for these rubrics , although informal comparisons between children , primitive races and even primates and animals persisted . instead , the post - world war i professionalisation of american psychology led to a narrowing of scope from the domain of cognitive process to that of cognitive performance and ability. psychometric tests with industrial and military applications proliferated , often employing drawing tasks that extended child study techniques for circumscribing subjectivity to adult test takers . the manual nature of these tests , the inscriptions they produced and the systems of evaluation that psychologists developed helped to naturalise a set of cultural assumptions about correct ways of drawing , making them a proxy for healthy ways of thinking . of course , the possibility of individualised meaning and interpretation was not excluded from all areas of psychology that employed drawing as a technique . many practitioners understood patient art as a pathway to the otherwise inaccessible interior of troubled psyches . patient art figured prominently in the case studies of freud , jung and followers of psychodynamic psychology . this analytical approach to drawing filtered into american popular culture as the cult of the doodle that arose in the 1920s and 1930s , wherein armchair analysts collected the doodles of the rich and famous and subjected them to psychobiographical interpretation . whether freudian or popular , this interpretive school linked drawings to individual subjects and specific sociocultural settings . practitioners still , however , assumed that the relay between mind and hand bypassed the subject s conscious mediation a mechanism that allowed subliminal desires to be manifested in the idle margin scrawls of patients , presidents and movie stars . this was the same assumption that allowed drawing to serve child study so well . it became embedded in the technique , even when that technique served differing ends . this gives us one history of the emergence of drawing as a material research practice around the turn of the twentieth century . child study understood drawing as revealing the workings of the mind on a stratum both evolutionarily and developmentally prior to that of language . primitiveness helped it stand as objective evidence within a recapitulationist framework , obscuring the larger cultural context of art education and the specific context of individual subjects . hall s concept of the child mind was a taxonomic one , and thus drawings were taken as quasi - archaeological artefacts that captured normalised developmental stages . such a concept lent itself to application on a mass scale , as the evaluation of images was based on formal criteria correspondence with the conventions of representational drawing rather than on subjective content . the position of psychology in the early twentieth century was such that practical applications in psychometry and intelligence testing were highly valued as routes to professional legitimacy and funding . on the other hand , lukens and those who leaned towards interpretation believed that children s drawings give us one of the surest ways with which to reach the contents of their minds ; the meaning of images lay in the subjectivity of their makers . this inclination to interpret rather than evaluate , and to read drawings as expressions of complex subjectivity , saw its realisation in freud s wolf man drawings , the art therapy of margaret naumburg and melanie klein s child psychoanalysis in which drawing served as a paper tool. although psychoanalysis approached the mind from a very different perspective to psychometry , both envisioned the drawing child as a circuit through which the mind s contents flowed relatively unencumbered , an assumption facilitated by the earlier work of child study . by the early decades of the twentieth century , a variety of practitioners embraced the notion that drawings reveal hidden information about the mind , but disagreed as to whether that information pertained to the mind s contents or its function . the second and third cases presented here follow the functional strain of this divergence , showing how the exclusion of subjective content became especially important for researchers making new and controversial knowledge claims in psychology . at the same time as child study took up drawings as psychological data , they became central to another fledgling field devoted to the investigation of the human mind . this field , psychical research , developed a different experimental methodology around the same techniques of pencil and paper . like the practitioners of child study , psychical researchers reproduced drawings as material proof of an elusive type of mental activity , in this case , telepathic communication . just as drawings could reveal a developmental resonance between the mind of an individual and the collective mind of a race , they could also demonstrate psychic resonance between two minds . psychical researchers who studied telepathic phenomena employed drawings as evidence , portraying them as thought - pictures that manifested , in objective material form , the otherwise - concealed mental processes of mediums . even critics aiming to debunk telepathy used drawing to prove alternate explanations of the phenomenon ; they argued for the prevalence of certain images in an unconscious community of thought , the contours of which they empirically determined by collecting drawings from the population . child study emerged concurrently with the first major surge of interest in psychical research , and thus , my comparison is not meant to suggest a chronological relationship in which drawing tests were passed from one to the other . both fields sought to establish drawing as a straightforward instrument that accessed and recorded the contents of the mind . both understood drawings as material evidence of thought processes . and both attempted to exclude the individualised , subjective meaning of images from their analyses by placing particular kinds of subjects into controlled experimental settings . i position child study first in this essay because of its direct connection to the mid - nineteenth - century theories of pedagogy that established drawing as a universal human faculty with objective standards . mandatory art instruction established drawing as a conventional communicative mode shared by scientists , subjects and the public at large . a professionalisation story would proceed from psychical research on the margins of science to more respectable child study , and thence to scientific neuropsychiatry . i have attempted to tell a story not about professionalisation , per se , but about changing attitudes towards subjects and subjectivity . starting with child study sets the stage for a process of circumscription that did not proceed linearly or uniformly across the discipline . further , this process generated popular offshoots that held intense interest for the public and continually smuggled subjectivity back into the psychological interpretation of drawings . interest in mind reading and thought transference surged in anglo - american culture of the 1870s , in an environment suffused with spiritualist sances , mesmeric stage performances and other supernormal manifestations . the public was caught up in the fascination of the unusual to the great concern of sceptical scientists , who were often called upon to expose the tricks of psychic charlatans . historians assign this occult revival a dual meaning : it reflected an anti - materialist backlash against secular science , which threatened cherished beliefs about the immortality of the soul . at the same time , it proclaimed the progress of science in conquering the realm of the invisible . the many proposed naturalistic explanations for telepathy worked by analogy to the transmission of light , magnetism and electricity . if those intangibles could be measured and quantified with the new instruments of physics , it seemed plausible that human thought , too , might have its own yet - to - be - discovered wavelength and might travel from mind to mind like a telegraph message . in an age when communication technology had broken down the barriers of time and distance , and scientists were working towards the wireless telegraph and radio , such expectations were hardly far fetched . indeed , the psychical researcher and outspoken spiritualist oliver lodge performed what many claim was the first public demonstration of wireless telegraphy at a meeting of the british association for the advancement of science in 1894 , proving that ideas , in the form of electrical impulses , could leap through space . psychical researchers hoped to determine whether impressions from the minds of those about us [ can reach ] our own minds by channels distinct from those of the senses. this was the widely adopted working definition of the phenomenon alternately named thought transference or telepathy . although they saw their project as empirical , researchers faced the challenge of studying people who claimed that their powers of mediumship or clairvoyance appeared only under very particular conditions . peter lamont has pointed out that psychic practitioners and sceptical inquirers almost never agreed on the evidential criteria for psychic phenomena ; their disagreements , however , were quite nuanced , as both claimed to be working in an empirical , evidence - driven paradigm . the production of evidence in settings that blended the scientific , social and performative made for minute wrangling over the limits of observation and self - report . both public mediums , who received payment for stage shows , and private mediums , who demonstrated in parlours and sitting rooms for small circles of invited guests , combined popular spiritualism in the form of channelling and automatic writing with parlour games such as guessing cards or locating hidden objects . impartial scientific observers , but they shared a social context that made certain behaviours unacceptable . for instance , spiritualist publications criticised harvard psychologist hugo mnsterberg for an incident in which he took credit for grabbing the foot of the medium eusapia palladino to catch her cheating . palladino s supporters argued that such breeches of conduct endangered the medium , who was already in a fragile mental state from her exertions . psychical researchers regularly exposed performers tricks , but they did so within a set of practices that positioned the medium as a particular kind of subject at the mercy of forces , whether spiritual or physiological , that he or she did not control . researchers attempted to use drawing , a technology of direct inscription , to bypass the epistemological tensions that marked their relationships with their subjects . such investigations first took an institutional form in 1882 with the founding of the british society for psychical research ( spr ) by physicist , inventor and professed spiritualist william fletcher barrett . the spr placed mediums and sensitives under experimental conditions aiming to disentangle fraud from genuine phenomena . one of their foundational experiments involved the brighton duo of george albert smith and douglas blackburn , whose successful transmission of images became a pillar of the spr s argument in favour of telepathy . the spr needed a more rigorous method to pre - empt deception , and they settled on drawing . in august 1882 , blackburn , a local newspaper editor , wrote to the spiritualist periodical light , claiming that george albert smith could read his thoughts with an accuracy that approaches the miraculous. smith , only eighteen years old at the time , had recently begun performing as a medium in brighton . he and blackburn colluded to promote his act , with blackburn serving as a confederate onstage at the same time as he published glowing reviews in his paper . blackburn would later claim that , as a service to science and to the public , he and smith decided to reveal how easy a matter it was to take in scientific observers and credulous spiritualists. the spr s committee on thought - transference , led by barrett , edmund gurney and frederic myers , responded to blackburn s letter with interest . myers and gurney were cambridge educated and independently wealthy ; they turned their philosophical and scientific training to establishing psychical research as an area of inquiry . during the autumn of 1882 , the three committee members conducted a series of tests to verify smith s powers . they began with the bread - and - butter demonstrations of thought reading : guessing of numbers , names and colours chosen by the experimenters , shown to blackburn and transmitted from blackburn to smith . these transmissions occurred , the spr promised , with no sound or movement of the lips of any one ; smith and blackburn were , however , allowed to hold hands . when the two men were placed in separate rooms their run of successes came to an abrupt halt , and blackburn complained of neuralgia caused by the strain of concentration . gurney and his colleagues allowed them to resume contact , and they produced more successful thought transferences . although the spr investigators appreciated that hand - holding could be a means of sending a coded message , their subjects asserted that it was also the channel through which their extra - sensory thought transference worked . in the interest of fairness , the investigators decided to alter the content transmitted , rather than altering the experimental set - up , to exclude the possibility of coded communication . gurney framed this methodological development with the question , how far do impressions of drawings or geometrical figures , inexpressible in descriptive words , admit of being transferred? the activity of drawing would become an instrument for fraud - proof thought transference in the eyes of the spr . on the second day of experiments , gurney , myers and barrett drew some image at random , the figure being of such a character that its shape could not easily be conveyed in words ... in order to meet the assumption that some code such as the morse alphabet was used by s. and b. they showed this image to blackburn , blackburn grasped smith s hands and smith sat down and reproduced the image on paper , sometimes while wearing a blindfold . smith s drawings , about as like the original as a child s blindfold drawing of a pig is like a pig , were nonetheless recognisable as intended to represent the original figure. this comparison with the rough , unpolished appearance of child art supported the spr s assertion of naivet on the part of the medium . as with child study , an objective concept was discernible through the garbled communication medium of the scrawl. as with child study , close attention to the signal s distortion figure 6:(a ) the committee s drawing ; and ( b ) smith s reproduction . ( third report of the committee on thought - transference. proceedings of the society for psychical research , 1 ( 1883 ) , 213 . ( a ) the committee s drawing ; and ( b ) smith s reproduction . ( third report of the committee on thought - transference. proceedings of the society for psychical research , 1 ( 1883 ) , 213 . the spr took smith s drawings as authoritative evidence of direct communication between minds , asserting that the burden of explaining these results rests upon those who deny the possibility of thought - transference. they made a point of reproducing the drawings in their journal , the proceedings of the society for psychical research , with the assurance that the whole series of figures ( nine in number ) are given in the accompanying plates , which are engraved from photographic reproductions , on the wood blocks , of the original drawings ( emphasis original ) . the technology of photographic wood - block engraving guaranteed that no foul play had intervened ; the direct , unmediated proof was plainly visible , and circulated in the persuasive form of a scientific journal . wood - block engraving , the illustration technique that generated the vast quantity of images demanded by nineteenth - century mass print , had transformed in the second half of the century from an interpretive art to a facsimile process . an engraver in the 1840s would have drawn or traced a copy of smith s figures onto the block , potentially smoothing over the rough , unsteady lines to distil a more correct representational image . by 1882 , smith s figures could be photographed , the film printed directly onto a wood block and this photographic block carved by a technician trained in mechanical exactitude . engraving became harnessed to photographic technologies whose rhetoric of verisimilitude made it possible for thousands of people all over the world to examine the same piece of evidence . photo - engraving enabled the reproduction of specific images from individual research subjects like smith : it preserved the errors and idiosyncrasies that constituted the actual proof of smith s telepathic power . as engravings went from idealised representations to exact , unmediated copies , readers could use figures in scientific journals such as the spr s proceedings and hall s pedagogical seminary in a new way . gurney , myers and barrett described the act of telepathic drawing with terms like deliberate and continuous that suggested the automatic nature of the medium s activity ; smith worked as if copying a drawing that is seen. the resulting artefacts could be used to reconstruct the process that produced them . this method was attractive because the standardised objects cards , colours , numbers that more statistically inclined psychical investigators hoped to employ for scientific data collection offered little insight into the cause or nature of thought transference . were thoughts transmitted in the form of words , images or as pure concepts ( what william barrett termed smith s success in reproducing drawings inspired the spr to move forward with these interesting questions of how and why , using visual specimens to make their arguments to colleagues and readers . figure 7:(a ) the original drawing shown to blackburn ; ( b ) smith s first attempt at reproducing the drawing ; and ( c ) smith s second attempt at reproducing the drawing , after blackburn looked at the original again ( source : the third report of the committee on thought - transference. proceedings of the society for psychical research , 1 ( 1883 ) , 207 ) . ( a ) the original drawing shown to blackburn ; ( b ) smith s first attempt at reproducing the drawing ; and ( c ) smith s second attempt at reproducing the drawing , after blackburn looked at the original again ( source : the third report of the committee on thought - transference. proceedings of the society for psychical research , 1 ( 1883 ) , 207 ) . how of thought transference , the spr would have to disentangle perception from cognition and determine whether thought travelled as image or idea . after an apparently unsuccessful thirteenth trial , the spr investigators asked blackburn , the agent , to reproduce the original image that he had just seen and communicated telepathically to smith ( figure 7(a ) . blackburn s reproduction revealed that he had misremembered the original image , and communicated that incorrect image to smith ( figure 7(b ) . for the previous two decades , physiologists had debated whether visual perception was more dependent on processes within the eye or on psychical researchers saw an opportunity to weigh in on this question by accessing the mind as it pivoted between perception and cogitation . the main errors of mr smith s drawings existed already in mr blackburn s recollection of the drawing , they reported , demonstrating that smith received the inaccurate image from blackburn s flawed memory rather than as a purely visual sense impression . when the investigators showed blackburn the image again , and allowed smith to redraw it ( after grasping blackburn s hand ) , they attributed the improvement in smith s accuracy to blackburn s improved memory ( figure 7(c ) . these very specific uses of drawing accessed the workings of two minds that of the agent , blackburn , and the percipient , smith as they supposedly communicated a visual image . the spr s gurney , myers and barrett weighed the possibility of fraud and ruled it prohibitively difficult due to the inexpressible nature of the images . let our readers , who may be familiar with the morse or other code of signals , try in some such way to convey a description of one of our drawings , to a friend who is blindfolded and has not seen the original. drawing was such a useful instrument for the spr because it seemed to capture the irreducible nature of thought in a material form that could be scrutinised for evidence of mental processes . descriptions of the smith and blackburn experiments arrived in the united states in the winter of 18823 . american subscribers to the spr s proceedings , including william james , g. stanley hall , george s. fullerton of the university of pennsylvania and henry p. bowditch of harvard medical school , would have seen the report , and it received favourable notice in the scientific press as an eliminate[d ] every possible element of charlatanism. by the following year , with william james leading the charge for an autonomous american version of the spr , the prospective leaders of this group began critically re - evaluating the british society s findings . although the american society represented itself as a sceptical counterweight to the spiritualist leanings of the british , their reviews of the smith and blackburn report embraced the use of drawing as an experimental methodology . in the published text of his first presidential address to the american society for psychical research ( aspr ) , circulated in the proceedings of july , 1886 , simon newcomb focused his critique on the spr s inconsistent procedures . in the experiments with smith and blackburn , smith was sometimes blindfolded , sometimes not ; sometimes smith and blackburn spoke to each other , conditions changed constantly as the experimenters thought of new scenarios they wanted to test . whereas gurney , myers and barret asserted that our experiments derive much strength and coherence from their very multitude and variety , newcomb regarded these conditions as unacceptably haphazard : without rigid controls , we have no right to attribute the result to one cause [ ie . , telepathy ] rather than another. this tension between the rigid planning and record keeping of the laboratory and the curiosity - driven exploration of the gentleman or amateur investigator would characterise the aspr s relationships with many of its own constituents , as well as with its british counterpart . the actual drawings that smith produced represented the most stable , scientifically valid element of the entire undertaking in newcomb s assessment . even in less - than - ideal conditions , these copies of drawings have a great advantage over verbal descriptions , newcomb noted , in that the record can be made the subject of future study. the permanent , reproducible nature of images was an antidote to the anecdotal style that newcomb and his colleagues found most galling in the spr s work . interestingly , newcomb s published endorsement of the smith drawings represented a change of course from the more suspicious note that he sounded in his actual presidential speech , on 12 january 1886 . in the speech itself , he deemed smith s drawings fraudulent . what changed his mind was a new set of sketches that he exchanged with william james in the months after his speech , through which james demonstrated the possibility of non - supernatural blindfolded draughtsmanship . your drawings seem to show that ... the [ smith ] drawings in question were at least possible , newcomb wrote to james on 16 february . their correspondence reveals the extent to which the practice and material product of drawing served as a persuasive tool for leaders of the aspr . while newcomb doubted the circumstances of the british society s procedure , a trustworthy colleague such as william james could produce corroborating evidence from his own hand . thanks to an efficient postal service and the portable nature of drawings , newcomb could then inspect the evidence first hand . their disagreement was resolved through the mail over the course of a mere ten days , and newcomb edited the text of his speech to reflect his confidence in drawings as evidence . the aspr further endorsed drawing as a methodology in 1885 , urging its members to attempt the experiments which have attracted so much attention from the english society. this represented an exciting expansion of their research program beyond the repetitive experiments requested in the society s previous circulars , such as the guessing of numbers , colours and cards . the society acknowledged the heightened appeal of personalised and infinitely variable drawing tasks : as the experiment of free - drawing may prove more interesting to some persons , it is hoped that more elaborate figures may be tried , and the results forwarded. figure 8:illustration from thought - transference by means of pictures. source : proceedings of the american society for psychical research , 1 ( 1885 ) , 44 . illustration from thought - transference by means of pictures. source : proceedings of the american society for psychical research , 1 ( 1885 ) , 44 . william henry pickering , a harvard astronomer and founding member of the aspr , published the results of such an experiment as a model accompanying this call for participation . pickering s drawings were made on one or two evenings as an amusement among friends , with a female acquaintance acting as the sensitive , so called. like most psychical experiments , these were a parlour activity , but one with greater entertainment value and greater power as evidence than the mundane and easily fudged guessing tasks . the graphic summary of pickering s experiment is particularly striking : all fifty - two pairs of drawings were reduced to thumbnail size and condensed into one page . this public presentation of a complete run of experiments asserted the scientific transparency of the aspr in contradistinction to the spr , which mainly printed examples of its successful results . the miniature images conserved valuable pages , but also conveyed the impression of a complete formal taxonomy rather than an informal parlour game . an aggregate view de - emphasised the idiosyncrasies of subject and circumstance that the spr used to justify the failures and fine tuning of their procedures . such serial presentation had become an important tool of visual rhetoric for nineteenth - century sciences ranging from physics to embryology , including pickering s discipline of astronomy , where debate over the nebular hypothesis in the 1830s had centred around the arrangement of successively brighter nebulae into an order that suggested temporal progression from diffuse clouds of matter to stars . pickering s grid of images , lacking any apparent order or progression , subtly asserted the absence of theory - driven manipulation and modelled the randomness necessary to ensure impartial psychical experimentation . drawings and diagrams featured regularly in the first year of the aspr s proceedings , but , for sceptics within the organisation , this visual evidence soon lost the lustre of the early smith and blackburn successes or rather , it was appropriated by a different camp within psychical research as proof against , rather than for , the reality of thought transference . gurney , myers and barrett of the spr interpreted resemblances among drawings as evidence of communication demonstrating direct transmission of content between minds . when simon newcomb embraced drawings as evidence , he cited the possibility for reinterpretation as their main advantage : they could be made the subject of future study. as the aspr set about gathering drawings from its own far - flung correspondents , some began to view the mass of responses as indicating unconscious habits or statistical probability rather than telepathy . such complex explanations were ultimately not necessary to account for the smith and blackburn phenomenon that had established telepathic drawing as a preferred methodology in psychical research . in a confession published in the popular magazine john bull more than twenty years after his experiments with smith , blackburn explained that he had used a numbered ten - by - ten grid to reproduce the transferred images , while smith indicated coordinates on the grid through hand squeezes , breathing or tapping the carpet . this method yielded drawings with an overall resemblance in shape , although smith often failed to recognise ( or intentionally botched ) the referential content of the original picture . interestingly , the use of a grid for proportion and perspective drawing was also part of art pedagogy , but faced criticism precisely on the grounds that it created superficial likeness without teaching the skills of active , synthetic observation . the spr s committee on thought - transference had acknowledged the possibility that smith and blackburn were using a secret code , but expressed confidence in their assessment of the men s characters and in the difficulty of cheating . it is probably no exaggeration to say that several scores , if not hundreds , of precise signs would be required to convey an idea as exact as that conveyed in many of mr smith s representations. indeed , a widely accessible popular literature for aspiring thought - readers provided systems for organising and communicating hundreds of precise signs . it is likely that smith and blackburn were familiar with such pamphlets , often attributed to famous stage performers such as w. irving bishop and robert houdin . figure 9:(a ) an alphabetical grid for sending images in the form of a code ; and ( b ) calostro gives an example of how to use the grid to transmit the image of a house . source : calostro ( pseudonym ) , the radio vision mind - reading code ( closter , nj : calostro publications , 1930 ) , 32 . ( a ) an alphabetical grid for sending images in the form of a code ; and ( b ) calostro gives an example of how to use the grid to transmit the image of a house . source : calostro ( pseudonym ) , the radio vision mind - reading code ( closter , nj : calostro publications , 1930 ) , 32 . the spr quietly dropped the smith and blackburn case from their canon of evidence , but the efforts of both the british and american societies to elicit telepathic drawings from the public had by then established this methodology among amateur researchers who understood it on their own terms , and adapted it to their changing interests . from 1904 until the 1940s , french parapsychologist ren warcollier worked with a method similar to the spr s , in which an agent observed a target image and distant percipients tried to draw it . warcollier s remote viewing experiments were remarkable for their scale and global scope , involving much like the spr investigators , he was not searching for perfect matches , nor for insight into his subjects psyches . he believed that analysing discrepancies and resemblances between agent and percipient drawings would reveal the mechanisms of mind - to - mind communication . by 1930 , when the novelist upton sinclair published a book of his telepathic drawing experiments entitled mental radio , the popularisation of freudian theory had opened up a wide range of new interpretive possibilities for mental imagery . yet sinclair largely hewed to the mode of analysis represented by the spr and warcollier , comparing the visual appearance of drawings and sometimes taking into account their manifest content ; only in a few isolated cases did he note the potential impact of the subject s history or emotions on a transmission . psychical researchers were engaged with the surface level of drawings , looking at their geometric and linear components and the explicit objects or ideas that they represented . like the collectors of children s drawings , they seemed increasingly nave in their approach from the perspective of psychologists who were either unpacking or working to contain the problem of subjectivity . after the deflation of the spr s case for telepathy in the first decade of the twentieth century , sceptical psychologists came to expect that , like smith and blackburn and any number of supposed psychic mediums , adults brought complex motives and resources to bear on drawing tasks . understanding these motives would require more art than science , and thus was not particularly useful in the goal of gaining scientific legitimacy for psychical research or for laboratory psychology in general . psychoanalysis threatened the integrity of drawings as automatic traces of mental processes by throwing open the gates of interpretation . however , there remained a special kind of subject whose neuropathologies produced characteristic changes in cognitive function that the act of drawing could make visible . simple black - and - white line drawings floating on a page of scientific text , labelled with a figure number and caption , helped to define a new area of neuropsychology in the mid - twentieth century . drawings became evidence of the internal workings of cognitive mechanisms not accessible through more holistic methods of testing and self - report . neurologists had used patients drawings to study the mechanics of cognition since the 1870s , slowly embracing the notion that these drawings , if elicited under controlled conditions and analysed by the appropriate experts , reveal how localised damage impacts specific pathways in the brain . neuropsychological literature has typically depicted patients with brain injuries in terms of their cognitive functions , or lack thereof , and , within this literature , patient art is presented as the output of damaged circuits rather than as subjective expression , in much the same way that child study and psychical research carefully circumscribed the agency of their drawing subjects , preferring to depict them as self - recording devices . the german physiologist herman munk is credited with coining the term psychic blindness or mind - blindness , in 1881 based on experiments in which he cauterised specific areas of the cortex in dogs . dogs with posterior occipital lesions were unable to identify their food bowls or recognise their handlers , although their vision was intact . in 1888 , the neurologist heinrich lissauer examined an eighty - year - old patient , mr gottlieb l. , suffering from a strange array of perceptual problems similar to those displayed by munk s dogs : the patient could see things but could not recognise them , unless allowed to examine them by touch . he could draw simple copies of pictures shown to him , but could not name the objects represented . lissauer used drawings to probe his patient s visual processing deficits because verbalization may not be accompanied by visual imagery ... one can get much further by asking people to draw from memory the same reasoning given by lukens in studying children s conceptual knowledge . based on his extensive work with gottleib l. , lissauer postulated that psychic blindness resulted from damage to the occipital areas of both hemispheres that prevented the association of visual perceptions with knowledge stored in memory , a condition that sigmund freud , still practising neurology in 1891 , termed agnosia , or loss of knowledge. the concept of agnosia that damage along its processing and integration streams can prevent the brain from connecting sensory input with meaning raised the possibility of physiologically divorcing vision from comprehension . a patient with this class of disorder a canny diagnostician could use the machine s output to locate the point at which normal visual processing had broken down . interest in cognitive deficits caused by localised neurological injury , specifically in the disorders of language processing called aphasias , surged in the 1850s , as neurologists such as paul broca and carl wernicke pursued the localisation of functions by correlating patient histories with autopsy findings . broca , wernicke and their peers debated the philosophical implications of language localisation ; aphasia became a way of probing what defines the human and what constitutes a conscious self . these high - profile aphasia debates are well studied , but few historians of science have engaged with the more recent history of agnosias , beginning with lissauer in the 1880s . unlike aphasia , which was manifested overtly in verbal communication , agnosias were slippery and difficult to observe in the clinical settings of the late nineteenth century . as lissauer noted , accessing patients perceptions required a technique not dependent on language or self - report . historians have , perhaps , been attracted to nineteenth - century aphasiology because its disruption of the correspondence between language and reality resonates strongly with late twentieth - century disruptions in the discipline of history itself . agnosia engages more recent historiographic trends , allowing us to extend the narrative of how science has mobilised visual evidence to depict brain functions untethered from human subjects . while aphasias are specific to the processing and production of language , agnosias encompass broader sensory deficits ranging from impaired face recognition ( prosopagnosia ) to impaired sensory perception of one s own sensory perception disorder ( anosognosia ) . classification of these disorders has , as researchers inevitably note in their literature reviews on the subject , depended on the availability of patients with highly specific lesions , a rare occurrence given the diffuse damage normally caused by head injury or stroke . historically , a contingent of neuropsychologists have made the case that categorisation is a useless exercise due to the individualised nature of each patient s difficulties , and that particular classes of agnosia do not exist as clinical entities . in this debate , drawing once again became a decisive tool and evidentiary form . it helped researchers develop an etiology of agnosias ; the examples given here centre on visual agnosia , but drawing became almost ubiquitous in assessments for a wide range of interrelated sensory perception and processing disorders . figure 10:a drawing task with original images on the top line and the patient s copies below . source : w. russell brain , visual object - agnosia with special reference to the gestalt theory. brain 64 ( 1941 ) , 48 . a drawing task with original images on the top line and the patient s copies below . source : w. russell brain , visual object - agnosia with special reference to the gestalt theory. brain 64 ( 1941 ) , 48 . tests like that shown in figure 10 , in which a patient was asked to copy three geometric forms , seem to project the subject s faulty perceptual function directly onto the page in a way that requires little interpretation . however , such images are rich with theoretical assumptions and rhetorical strategies that reinforce the immediacy of the drawing as a materialisation of mental processes . a set of characteristic drawing tasks emerged in european and american clinical neurology in the early twentieth century that relied on a shared visual repertoire of mundane objects , clocks , maps , geometry and perspectival rules . some newly diagnosable cognitive disturbances were subtle enough that patients compensated for them in everyday life without overt signs of difficulty . dysfunction was present , however , if a patient was quietly deviating from the normal pathways of image recognition and production built into the cultural practice of drawing . british neurologist henry head , in his authoritative 1926 work on aphasia , developed four drawing tasks for evaluating the nature of a patient s cognitive disability : drawing from a model , drawing from command ( head generally requested an elephant ) , drawing the ground plan of a familiar room and drawing anything that [ comes ] to mind. each of these tasks assumed a shared visual grammar rooted in western art and art education . jacyna , in his biography of head , underscores the importance of head s position at the empire hospital in vincent square during world war i , where he treated young , well - educated officers who had suffered acute head trauma . their social backgrounds and the specific nature of their battle wounds made these patients ideal for isolating pure forms of aphasia and agnosia . whereas their presumed naivet made the contents of children s minds easy to capture in drawings , the presumed intellectual soundness of head s patients prior to injury meant that the injury itself was the thing captured in diagnostic drawing tests . head reproduced their drawings as the only illustrations , aside from x - rays , in his two - volume tome . source : henry head , aphasia and kindred disorders of speech ; ( cambridge university press , 1926 ) , 131 ; and ( b ) patient no . source : henry head , aphasia and kindred disorders of speech ; ( cambridge university press , 1926 ) , 131 ; and ( b ) patient no . 25 s head is a prominent example of how neurologists in clinical settings developed ad hoc drawing tasks for the evaluation of patients with agnosic symptoms , using logic to connect errors in representation with localised brain damage . head attempted to create a more generalised set of evaluation criteria , applying a larger number of tests to a larger number of patients than previous researchers had . until this point , most agnosia research , like the smith blackburn experiments , was performed on isolated patients under test conditions that varied depending on the patients responsiveness and abilities . despite head s attempt to impose a standardised diagnostic framework , there was no professional consensus on the disease categories that various drawing tasks identified until the 1970s . in the 1940s , edinburgh neurologists oliver zangwill and andrew patterson developed a visual testing battery for patients suffering from spatial disorientation . by asking patients to bisect lines , copy drawings and mark different times of day on a clock face , they connected the recognised phenomenon of unilateral neglect with specific disruptions in processing caused by unilateral parietal lesions . their testing battery was widely adopted , and their explanation of unilateral neglect supported a modular view of brain function that would gain increasing acceptance in subsequent decades , building on this model of matching a lesion to a deficit revealed through drawing . drawing tasks helped researchers distinguish the discrete cognitive operations contributing to patients broader deficits , and refined these cognitive operations into successively narrower categories . throughout the 1950s and 1960s , a group of neuropsychologists argued that cases classed as agnosias did not reflect a specific disorder , but rather resulted from generalised brain damage and decreased cognitive functioning . to my knowledge of the literature , there does not exist a single case of agnosia without elementary sensory disorders and without mental deterioration , claimed german neurologist eberhard bay . bay and his supporters insisted that there is no need to search for any mystical gnostic activity to be disturbed , a point that sometimes led to questioning the intelligence or integrity of agnosic patients such as schn , a world war i veteran whom bay accused of exaggerating his symptoms . debates over the reality of this syndrome were particularly fraught because clinicians also played an advocacy role for their patients , certifying the need for pensions or disability support . advocates for visual agnosia as an etiologically distinct syndrome used drawings as evidence for a new theory of visual processing , modelling it as a stream that flowed from lower - level perception to stored perceptual knowledge to stored conceptual knowledge . along with a battery of other visual tasks , drawings helped to delineate a disorder which gained widespread acceptance with the work of george ettlinger , glyn humphreys and jane riddoch from the late 1960s onwards . dementia is a convenient label to apply when aspects of behavior are not fully understood , wrote humphreys and riddoch ; for agnosic patients , having a problem identified and labeled can be a reassurance. drawings captured patients subtle and disorienting errors in spatial representation , assembling parts into wholes , or recalling the forms of common objects , fixing in material form specific deficits that had once seemed diffuse and intractable . drawing tests mobilise an arsenal of familiar objects depicted in outline from a conventional point of view , in part , as an accommodation to the perceptual difficulties of neurological patients . pictures are much more difficult [ to recognise ] if the artist has used shade , reflection , or the skills of impressionism , reported john , the patient studied by humphreys and riddoch . this visual vocabulary also dispenses with the subjective problem of artistic expression , aiming , instead , for a conventional sense of accuracy shared by patients and researchers . i never had much drawing ability or the simplest comprehension of how to show a third dimension in scribbling [but ] i can comprehend enough of my own handiwork to know if it is a reasonable representation of what i had in mind. john s ability to produce a drawing and then reflect on its correspondence with an objective reference point acts as an assurance that he has no special training or expressive intentions he is a subject appropriately constrained in his use of drawing as an extension of pure cognitive function . batteries of drawing tests helped clinicians order lesions by location and construct a theory of the processing stream , in the same way that ordering children s drawings along a developmental sequence enabled psychologists to construct an evaluative framework based on that sequence . throughout these debates , neurologists recognised that deficits and lesions rarely match in a simple one - to - one correspondence because injuries are rarely limited to a single - function area of the brain ; patients often suffer from a combination of difficulties following an injury . however , once the disorder agnosia emerged as a medical category , neurologists could use the techniques developed in establishing the diagnosis to disaggregate symptoms and locate damaged points on the visual processing stream . this systematic use of patient drawings in an experimental context to illuminate fundamental cognitive processes rests on the same material substrate as the spr s experiments with smith and blackburn and g. stanley hall s catalogue of the child mind . though not an intentional continuation of these practices , it reveals how the epistemological assumptions built into supposedly basic psychomotor tasks have shaped different areas of psychology in similar ways . drawing seemed a reliable means to capture the mental processes of particular kinds of subject while bypassing the unruliness of subjectivity . in each of the examples above , researchers identified subjects who they believed could function as object - instruments capable of faithfully recording the desired mental phenomena . nave children , blindfolded mediums and head trauma victims , equipped with pencil and paper , could act as their own soul catchers in the sense of a material assemblage that fixes an ephemeral quality of the mind in a physical form . however , the goal of this process for researchers was to take what is conventionally understood as the soul out of the picture , leaving a pure mental function traced in black and white . much of the literature on scientific illustration and objectivity focuses on the production of images by experts , whether scientists , technicians or artists , disciplined to act as transparent recording media for the phenomena they observed . the subjectivity of the image maker is not intended to be part of the image s scientific meaning , although , of course , it is integral to the social and cultural construction of that meaning . on the other hand , certain image - making practices in psychology are understood to externalise important clues about the patient s subjectivity that can be interpreted for purposes of diagnosis and therapy . the images of mental function and dysfunction discussed above occupy a strange intermediate space . they did not illustrate observed phenomena , but rather manifested hidden mental phenomena in visible form . nor were they interpreted for emotional content rather , their scientific validity depended on the exclusion of meaning , history and individual voice . researchers in vastly different contexts elicited these drawings aiming to make an objective study of subjectivity and to access the mind through the hand . the nuclear receptors comprise a family of transcriptional regulators involved in a wide variety of biological processes such as embryonic development , differentiation and homeostasis [ 1 , 2 ] . the family includes ligand - dependent zinc - finger transcription factors for steroid hormones , estrogens , thyroid hormones , retinoids , vitamin d and other hydrophobic molecules . in addition , several family members are ' orphan receptors ' for which ligands have yet to be identified . nuclear receptors have been assigned to six subfamilies on the basis of evolutionary studies . as the first member of the sixth subfamily , gcnf is also known by its systematic name nr6a1 . on the basis of homology and expression profile , the receptor has been given the alternative name of retinoic acid receptor - related testis - associated receptor ( rtr ) . transfection experiments reveal that gcnf can act as a constitutive repressor when binding as a homodimer to promoters containing a direct repeat dna element 5'-aggtcaaggtca-3 ' ( dro ) [ 7 , 8 , 9 , 10 ] . the mouse receptor ( mgcnf ) is highly expressed in the developing embryonic nervous system and the labyrinthine layer of the placenta [ 12 , 13 ] . in the adult , high transcript levels are restricted to the developing germ cells [ 5 , 14 , 15 , 16 ] . northern analysis reveals a transcript of 7.5 kilobases ( kb ) in somatic cells and an additional message of approximately 2.4 kb in male germ cells . this size difference is at least partially due to different polyadenylation sites , and it is therefore assumed that both transcripts code for identical proteins of 495 amino acids . the protein sequence is encoded by 11 exons . when differentiation of p19 embryonal carcinoma cells is triggered by retinoic acid , the transcript and the protein are temporarily upregulated and then downregulated . isolation of a human cdna coding for a protein ( hgcnf ) with an identity to the mouse protein of 98.7% , similar regulation in mouse p19 cells and in the human embryonal carcinoma cell line nt2/d1 , together with the presence of two mrnas of approximately 7.5 and 2.2 kb in human testis , suggested similar functions for mouse and human gcnf [ 18 , 19 , 20 ] . the cloning of human cdnas that give rise to different hgcnf isoforms , however , suggests a higher complexity in humans . currently , four different hgcnf cdnas have been isolated that code for isoforms ranging in size from 454 to 480 amino acids . we have investigated the genomic structure of mammalian gcnf to determine how the different gcnf isoforms are generated . here we compare the exon / intron structure of the previously characterized mouse gene with the human ortholog . our study shows that alternative splicing generates at least three of the different gcnf isoforms . to understand how the different human gcnf mrna isoforms are generated , we have identified all human protein - coding exons . the alignment of the full - length human gcnf cdna ( genbank accession number s83009 ) with the genome sequencing data at the ncbi localized the first protein - coding exon on chromosome-9-derived working draft sequence element nt_008491 . the genomic sequence was aligned with the previously identified mouse exon 1 containing the putative translational start site . the comparison was extended up to position -100 with respect to the mouse cdna reaching farthest in the furthest 5 ' direction . in addition , 100 base - pairs ( bp ) of the first identified intron were included ( figure 1 ) . while the transcriptional start sites of gcnf are still elusive , the 5 ' ends of the first protein - coding exons can not be defined . with respect to the sequence in figure 1 , the furthest 5'-reaching human cdna ( s83309 ) starts with nucleotide 171 . the putative translational start codons are in positions 346 and 350 of the mouse and human sequences , respectively . these start codons are present in all full - length mammalian gcnf cdnas characterized so far , suggesting a common amino terminus for the different gcnf isoforms . the comparison of the mouse 5'-untranslated sequence with the human genomic dna reveals high conservation with identical sequence elements of up to 50 nucleotides . the presence of 18 cg dinucleotides conserved between human and mouse is suggestive of a regulatory function of the untranslated sequence . five different human cdnas with alternative 5 ' ends have , however , been reported to genbank ( s83309 , u80802 , af004291 , nm001489/u64876 , x99975 ) . a comparison with the genomic sequence ( nt_008491 ) shows the sequence variation ( figure 2 ) . single - nucleotide polymorphism among cdnas isolated from different human libraries may reflect variants in the human population . two cdnas ( u64876/nm001489 , x99975 ) differ in their untranslated region with respect to the genomic sequence . therefore , it can not be ruled out that these cdna ends may have been generated during the cloning process . in addition , one of the cloned cdnas , coding for hgcnf-3 ( af004291 ) , has a deletion in the coding region of the first exon , giving rise to an open reading frame of 454 amino acids . the 5 ' part of hgcnf-3 has been isolated by the polymerase chain reaction , suggesting that this deletion may have been generated during the synthesis . the isolation of additional cdnas may give a clue as to which variants are true gcnf isoforms . the functional significance of the different isoforms is , at present , unknown but may lead to different transcriptional properties of gcnf isoforms . the comparison of the genomic sequences of exons 2 to 11 was extended by 100 bp of intronic sequence in both directions ( figure 3 ) . during the preparation of this manuscript all sequence information was made available by the international human genome project collaborators at the ncbi database and included in the contig nt_008491 . sequences of the 5'-untranslated region and of exon 7 obtained with a genome walking approach did not diverge from the sequence at the ncbi . two short exons of 42 bp and 45 bp , respectively , follow the first protein - coding exon in the mouse . short exons are relatively rare in mammalian genomes . the structure of the second protein - coding exon is conserved ( figure 3a ) . interestingly , on the basis of the genomic cdna , the third exon is highly conserved as well ( figure 3b ) . the human splicing apparatus preferentially , or exclusively , skips this putative exon , however . as splicing is highly regulated , a splice enhancer present in the mouse genome may not be present in the human genome . consequently , all known human gcnf isoforms lack the amino acids encoded by the putative third exon . of the 243 bp exon 4 that encodes the core of the dna - binding domain one of the reported sequences ( u64876/nm_001489 ) has a c to a transversion , however , which changes a codon for asparagine to one for lysine . splicing of exon 2 to exon 4 at the position characterized in the mouse results in isoform hgcnf-2 . in addition to this splice acceptor position , a splice acceptor site located 12 nucleotides further downstream is used to generate hgcnf-1 . two hgcnf-2 variants , hgcnf-2a and hgcnf-2b , which differ by a single amino acid , have been isolated . as speculated , alternative splicing generates the isoform 2b with a deletion of a serine residue . splicing to an acceptor site of exon 7 located three nucleotides the sequence and structure of exons 8 to 11 are also highly conserved ( figure 3g , h , i , j ) . the comparison of the 11th exon was extended up to the end of the human cdna sequence of s88309 . highly conserved sequence elements of up to 91 identical nucleotides indicate a regulatory function of the 3'-untranslated sequence following the translational stop codon . the aceview analysis at the ncbi based on the draft sequence and a blast search with s83309 of the celera genomics freely accessible whole - genome sequence data gave mostly similar intron sizes . both analyses revealed a large first intron of 37,652 bp in the public sequence data , and 37,157 bp in the private data . the size of the second intron separating exon 2 and exon 4 was only available in the ncbi database ( 14,869 bp ) . according to the ncbi and celera databases , introns 3 to 9 have sizes of 10,486 ( ncbi ) ( 10,471 , celera ) bp , 3629 ( 3615 ) bp , 190,321 ( 1708 ) bp , 1963 ( 1960 ) bp , 2716 ( 9019 ) bp , 1905 ( 1912 ) bp , and 1927 ( 1928 ) bp , respectively . the comparison of both analyses shows that the deduced sizes of two of the human introns differs greatly . it seems likely that these inconsistencies will be corrected in the final assembly of the human genome . to understand how the different human gcnf mrna isoforms are generated , we have identified all human protein - coding exons . the alignment of the full - length human gcnf cdna ( genbank accession number s83009 ) with the genome sequencing data at the ncbi localized the first protein - coding exon on chromosome-9-derived working draft sequence element nt_008491 . the genomic sequence was aligned with the previously identified mouse exon 1 containing the putative translational start site . the comparison was extended up to position -100 with respect to the mouse cdna reaching farthest in the furthest 5 ' direction . in addition , 100 base - pairs ( bp ) of the first identified intron were included ( figure 1 ) . while the transcriptional start sites of gcnf are still elusive , the 5 ' ends of the first protein - coding exons can not be defined . with respect to the sequence in figure 1 , the furthest 5'-reaching human cdna ( s83309 ) starts with nucleotide 171 . the putative translational start codons are in positions 346 and 350 of the mouse and human sequences , respectively . these start codons are present in all full - length mammalian gcnf cdnas characterized so far , suggesting a common amino terminus for the different gcnf isoforms . the comparison of the mouse 5'-untranslated sequence with the human genomic dna reveals high conservation with identical sequence elements of up to 50 nucleotides . the presence of 18 cg dinucleotides conserved between human and mouse is suggestive of a regulatory function of the untranslated sequence . five different human cdnas with alternative 5 ' ends have , however , been reported to genbank ( s83309 , u80802 , af004291 , nm001489/u64876 , x99975 ) . a comparison with the genomic sequence ( nt_008491 ) shows the sequence variation ( figure 2 ) . single - nucleotide polymorphism among cdnas isolated from different human libraries may reflect variants in the human population . two cdnas ( u64876/nm001489 , x99975 ) differ in their untranslated region with respect to the genomic sequence . therefore , it can not be ruled out that these cdna ends may have been generated during the cloning process . in addition , one of the cloned cdnas , coding for hgcnf-3 ( af004291 ) , has a deletion in the coding region of the first exon , giving rise to an open reading frame of 454 amino acids . the 5 ' part of hgcnf-3 has been isolated by the polymerase chain reaction , suggesting that this deletion may have been generated during the synthesis . the isolation of additional cdnas may give a clue as to which variants are true gcnf isoforms . the functional significance of the different isoforms is , at present , unknown but may lead to different transcriptional properties of gcnf isoforms . the comparison of the genomic sequences of exons 2 to 11 was extended by 100 bp of intronic sequence in both directions ( figure 3 ) . during the preparation of this manuscript all sequence information was made available by the international human genome project collaborators at the ncbi database and included in the contig nt_008491 . sequences of the 5'-untranslated region and of exon 7 obtained with a genome walking approach did not diverge from the sequence at the ncbi . two short exons of 42 bp and 45 bp , respectively , follow the first protein - coding exon in the mouse . short exons are relatively rare in mammalian genomes . the structure of the second protein - coding exon is conserved ( figure 3a ) . interestingly , on the basis of the genomic cdna , the third exon is highly conserved as well ( figure 3b ) . the human splicing apparatus preferentially , or exclusively , skips this putative exon , however . as splicing is highly regulated , a splice enhancer present in the mouse genome may not be present in the human genome . consequently , all known human gcnf isoforms lack the amino acids encoded by the putative third exon . of the 243 bp exon 4 that encodes the core of the dna - binding domain , 225 bp are identical in both species ( figure 3c ) . one of the reported sequences ( u64876/nm_001489 ) has a c to a transversion , however , which changes a codon for asparagine to one for lysine . splicing of exon 2 to exon 4 at the position characterized in the mouse results in isoform hgcnf-2 . in addition to this splice acceptor position , a splice acceptor site located 12 nucleotides further downstream is used to generate hgcnf-1 . two hgcnf-2 variants , hgcnf-2a and hgcnf-2b , which differ by a single amino acid , have been isolated . as speculated , alternative splicing generates the isoform 2b with a deletion of a serine residue . splicing to an acceptor site of exon 7 located three nucleotides the sequence and structure of exons 8 to 11 are also highly conserved ( figure 3g , h , i , j ) . the comparison of the 11th exon was extended up to the end of the human cdna sequence of s88309 . highly conserved sequence elements of up to 91 identical nucleotides indicate a regulatory function of the 3'-untranslated sequence following the translational stop codon . the aceview analysis at the ncbi based on the draft sequence and a blast search with s83309 of the celera genomics freely accessible whole - genome sequence data gave mostly similar intron sizes . both analyses revealed a large first intron of 37,652 bp in the public sequence data , and 37,157 bp in the private data . the size of the second intron separating exon 2 and exon 4 was only available in the ncbi database ( 14,869 bp ) . according to the ncbi and celera databases , introns 3 to 9 have sizes of 10,486 ( ncbi ) ( 10,471 , celera ) bp , 3629 ( 3615 ) bp , 190,321 ( 1708 ) bp , 1963 ( 1960 ) bp , 2716 ( 9019 ) bp , 1905 ( 1912 ) bp , and 1927 ( 1928 ) bp , respectively . the comparison of both analyses shows that the deduced sizes of two of the human introns differs greatly . it seems likely that these inconsistencies will be corrected in the final assembly of the human genome . in summary , our analysis reveals a conserved structure for gcnf , allows the verification and systematic analysis of splice variants , and may be the basis of a better understanding of gcnf . the conservation of the intron - exon boundaries is consistent with the extremely high degree of amino - acid conservation between the human and the mouse proteins . the generation of the proteins hgcnf-1 , hgcnf-2a and hgcnf-2b can be explained by alternative splicing of the rna . the sequence of the third coding mouse exon , including the splice sites , is highly conserved ; however , at present no human cdna has been isolated containing this putative exon . alternative splicing provides a plausible means for generating diversity and may contribute to a higher instructive complexity in human gcnf . exons of gcnf were identified by a blast search with the human gcnf cdna sequence ( s83009 ) in the " unfinished high throughput genomic sequences " and in the homo sapiens genomic contig sequences at the ncbi [ 24 , 25 ] . intron sizes given by the aceview analysis were compared with the numbers obtained by a blast search of celera 's assembled sequence of the human genome . the putative human gcnf exon 3 was identified by a blast search with the sequence of the third mouse exon . sequences were aligned using the wisconsin package version 10.0 of the genetics computer group ( gcg ) , madison , wisconsin . exons of gcnf were identified by a blast search with the human gcnf cdna sequence ( s83009 ) in the " unfinished high throughput genomic sequences " and in the homo sapiens genomic contig sequences at the ncbi [ 24 , 25 ] . intron sizes given by the aceview analysis were compared with the numbers obtained by a blast search of celera 's assembled sequence of the human genome . the putative human gcnf exon 3 was identified by a blast search with the sequence of the third mouse exon . sequences were aligned using the wisconsin package version 10.0 of the genetics computer group ( gcg ) , madison , wisconsin . the mouse - derived ( m ) dna sequence ( upper line , genbank accession number af254575 ) of the first protein - coding exon and flanking sequences are compared with the human - derived ( h ) sequence ( lower line , s83309 for the coding , and nt_008491 for the flanking sequences ) . identical nucleotides are highlighted in the human sequence by bold letters , gaps in the alignment are shown as dots . the deduced amino - acid sequence of the human protein is shown in the single - letter code . the bold p ( proline ) marks the position of an arginine in the mouse protein . sequence comparison of the 5 ' ends of human gcnf cdnas coding for a full - length protein with the genomic dna sequence . human cdna sequences have been aligned omitting the cloning sites at the 5 ' ends . one cytosine and five adenosines in the genomic sequence not found in any of the cdnas are underlined . the deduced amino - acid sequences ( aab50876 , aac52054 ) are given in the single - letter code . the upper line of nucleotide sequence shows the murine protein - coding exons and their flanking sequences ( af254815-af254821 ) . mgcnf indicates the deduced mouse protein sequence ; hgcnf indicates the deduced human protein sequences ; gcnf indicates identical protein sequences . forty - one out of 42 nucleotides are identical and the flanking splicing signals are conserved . a homologous sequence coding for identical amino acids was found in the human genomic sequence . the splice donor site shows the typical pyrimidine - rich sequence followed by the sequence 5'-ncag in both sequences , but the comparison reveals several base transitions . ( c ) a single splice donor site in the fourth exon coding for the dna - binding domain is used in all mouse - derived cdnas described so far . for the human isoform gcnf-2 , the corresponding splice site is used , giving rise to a protein containing the sequence isvsdd instead of the vsvpdd in mouse . usage of an alternative splice site located 12 bp further downstream gives rise to the shorter isoform gcnf-1 . an asparagine ( n ) is underlined because one of the human cdna clones codes for a lysine in this position ( u64876/nm_001489 ) . ( d ) sequences of the fifth exon coding for the carboxy - terminal extension of the dna - binding domain are highly conserved . an arginine in hgcnf-2b instead of serine results from alternative splice donor sites of the seventh exon . ( f ) the comparison of the seventh exon reveals three positions where the mouse and the human isoforms diverge . isoform hgcnf-2b is generated by using a splice donor site located three nucleotides further downstream . the exons coding for the putative -helices 3 to 6 ( g ) , 7 and 8 ( h ) , 9 and 10 ( i ) , 11 and 12 ( j ) in the ligand - binding domain are highly conserved . the comparison of the last coding exon in ( j ) was extended up to the end of the human cdna sequence of s88309 .

researchers in the mind sciences often look to the production and analysis of drawings to reveal the mental processes of their subjects . this essay presents three episodes that trace the emergence of drawing as an instrumental practice in the study of the mind . between 1880 and 1930 , drawings gained currency as a form of scientific evidence as stable , reproducible signals from a hidden interior . i begin with the use of drawings as data in the child study movement , move to the telepathic transmission of drawings in psychical research and conclude with the development of drawing as an experimental and diagnostic tool for studying neurological impairment . despite significant shifts in the theoretical and disciplinary organisation of the mind sciences in the early twentieth century , researchers attempted to stabilise the use of subject - generated drawings as evidence by controlling the contexts in which drawings were produced and reproduced , and crafting subjects whose interiority could be effectively circumscribed . while movements such as psychoanalysis and art therapy would embrace the narrative interpretation of patient art , neuropsychology continued to utilise drawings as material traces of cognitive functions . background : germ - cell nuclear factor ( gcnf , nr6ai ) is an orphan nuclear receptor . its expression pattern suggests it functions during embryogenesis , in the placenta and in germ - cell development . mouse gcnf cdna codes for a protein of 495 amino acids , whereas the four reported human cdna variants code for proteins of 454 to 480 amino acids . apart from this size difference , there is sequence conservation of up to 98.7% . to elucidate the genomic structure that gives rise to the different human gcnf mrnas , the sequence information of the human gcnf locus is compared to the previously reported structure of the mouse locus.results:the genomic structures of the mouse and human gcnf genes are highly conserved . the comparison reveals that the shorter human protein results from skipping the 45 base - pair third exon . three different human isoforms - gcnf-1 , gcnf-2a and gcnf-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon.conclusion:by homology with the mouse gene , 11 gcnf coding exons can be defined on human chromosome 9 . all human gcnf cdnas identified so far are , however , derived from mrnas generated by splicing the fourth to the second exon . although the genomic sequence is highly conserved , the analysis suggests that alternative splicing generates a higher complexity of human gcnf isoforms compared with the situation in the mouse .

we report involvement of unusual site in ilio - acetabular region with breach in articular cartilage . intra - operatively breach in acetabular roof was seen which was missed in the scan . this case report emphasizes about the rarity of the lesion in this location and the rare chances of breach in articular cartilage of the joint . bone desmoid tumor was an unknown entity until when jaffe hl reported a case in 1958 . it is a locally aggressive benign tumor reported commonly in mandible and meta - diaphyseal region of long bones . we report a case of desmoid tumor of ilio - acetabular region in a 40 year old female . a 40 year old female came with complaint of pain in the left hip region which aggravated on squatting and climbing stairs . radiological evaluation showed a lytic lesion in the left ilio - acetabular region , surrounded by a sclerotic margin superiorly . mri scan was done which showed a well defined homogenous lytic lesion with no break in the cortex and no soft tissue involvement ( fig . plain radiograph of pelvis with both the hips which shows a well defined lytic lesion over the left ilio - acetabular region . there appears no obvious breach in the cortex or the articular margin a ct scan of the pelvis was taken to rule out any breach in the cortex . there appears no obvious discontinuity through an ilio - inguinal incision the lesion was approached through inner table of ilium . a defect on the roof of the acetabulum was found which was missed by the scan ( fig . thorough curettage was done and the roof defect was reconstructed with a cortical graft harvested from the inner table of the ilium . there appears a hollow curetted lesion within which a breach in the roof of the acetabulum is seen histopathological finding showed multiple spindle shaped fibroblast cells with small and elongated nuclei in the background of dense collagen fibres . histopathology specimen picture showing spindle shaped fibroblast cells with small and elongated nuclei over a background of collagen fibers . there appears no nuclear atypia or mitotic activity patient was kept non weight bearing for 3 months with gradual return to full weight bearing walking . at follow up of 20 months patient is symptom free and xray showing no evidence of lytic lesion ( fig 5 ) . however , on ct scan there appears a persistent lytic area which we suspect to be a recurrence ( fig 6 ) . hence patient is kept on regular follow - up . a 20 month post operative x ray picture showing well maintained articular margin and the joint space . the lytic lesion seems filled up ct scan of the hip taken at 20 month post op , however shows lytic lesion anteriorly which seems to be a recurrence few cases have been reported since then and common occurrence being in mandible and meta - diaphyseal region of the long bone . recognition of desmoplastic fibroma is important because on radiology and histology , the lesion may be mistaken for an indolent , benign fibrous lesion or more aggressive spindle - cell sarcomas . it commonly occurs in the age group between the adolescent to 40 years with no specific sex predominance . patients commonly come with complaint of pain ; however there are few reported cases of pathological fracture especially those involving tibia and femur . the lesions are well defined with no sclerosis or periosteal reaction except if there is associated pathological fracture . if a soft tissue lesion is invading onto the bone , they are eccentric in position with sclerosis due to endosteal bone formation . significant t2 shortening of a non - sclerotic fibro - osseous lesion should place desmoplastic fibroma high among the diagnostic considerations . radiologically they have to be differentiated from giant cell tumor , fibrous dysplasia ( long bone lesions ) , fibrosarcoma , brown tumors , and chondromyxoid fibroma and hence a tissue diagnoses is must . the differentiation from a low grade fibrosarcoma is difficult though . as these are rare tumors , there is no standard protocol for treatment of these lesions . all kinds of procedures like intralesional , marginal or wide resection are reported as a treatment . but if wide resection would result a major functional deficit , an attempt of intralesional curettage with bone grafting seems warranted . desmoid fibroma of ilio - acetabular region is very rare hence a great sense of suspicion is required for the appropriate management . this case report emphasizes about the rarity of the lesion in this location and the chances of breach in articular cartilage of the joint . desmoid tumor , though rare , should be kept as one of the important differentials for the lytic lesion around pelvis . one should also define whether there is any associated cortical or articular margin breach ( as seen in our case report ) and then decide further management regarding reconstruction . uterine malformations can cause amenorrhea , infertility , recurrent pregnancy loss and pain and can also be normally functioning , depending on the nature of the defect [ 13,5 ] . the prevalence of uterine malformation is estimated to be 6.7% in the general population , slightly higher ( 7.3% ) in the infertile population , and significantly higher in a population of women with a history of recurrent miscarriages ( 16% ) . until the sixth week of life , the male and female genital systems are identical . there are two pairs of symmetrical genital ducts , the mesonephric ( wolffian ) ducts and the paramesonephric ( mullerian ) ducts . in the female embryo , the mullerian ducts grow caudally and become enclosed in the peritoneal folds , which later give rise to the broad ligaments of the uterus to which the ovaries , fallopian tubes and uterus are attached . the mullerian ducts approach each other and begin to fuse . at 9 weeks gestation , mullerian tract anomalies result from incomplete bilateral duct elongation , fusion , canalization , or septal resorption of the mullerian ducts . an accessory cavitated uterine mass ( acum ) is associated with an otherwise normal uterus . acum can be difficult to differentiate from true cavitated adenomyomas and cavitated rudimentary uterine horns . an accessory uterine mass can be caused by duplication and persistence of the ductal mllerian tissue in a critical area at the attachment level of the round ligament , possibly related to gubernaculum dysfunction . for the differential diagnosis of acum and cavitated non - communicating rudimentary uterine horns , hysterosalpingography showing a normal eutopic uterine cavity is helpful . an old classification had been put by jone h.w jr . in 1953 for the congenital anomalies of the female urogenital system : 1.uterus : ( a ) single uterus . a recent eshre / esge classification of female genital tract anomalies ( 1)uterine anomalies :- u0 . ( b ) without rudimentary cavity ( bi- or unilateral uterine remnants / aplasia ) . a 20-year - old female married for 2 years had undergone a full - term pregnancy with normal vaginal delivery , one year prior to this case report . the patient presented with 3 missed periods with lower abdominal pain , and a pelvic ultrasound examination revealed an intrauterine pregnancy of twins that had died at 13 weeks of gestation ( miscarriage ) ( fig . 1 ) . induction of abortion by oxytocin drip had been attempted twice but failed . the gynecologist proceeded to perform cervical dilatation and curettage , but the uterus was found to be empty . the patient was seen by another gynecologist who repeated the curettage and also did not find the dead twins , and an ultrasound after the procedure still showed the dead twin fetuses . the patient was then seen at our clinic , and we submitted the patient to another curettage . perforation of the uterus was expected ; thus , we opened the abdomen and found a slightly enlarged normally shaped and positioned uterus with 2 fallopian tubes and 2 ovaries . there was a uterine perforation at the fundus ; thus , the omentum was pulled in through the perforation , drawn out of the uterus , transfixed , ligated by suture and trimmed . the perforation of the fundus was repaired with 3 stitches using one zero vicryl sutures , no other visceral injury was found . on exploration , we found a large pear - shaped mass emerging from the right broad ligament with a thick pedicle that ended near the uterine cervix . because we did not obtain a detailed gynecological history of the patient , we assumed the mass was a broad ligament cyst . the mass was easily excised by clamping , transfixing , ligating and cutting the pedicle . the mass is pear - shaped and 10 13 cm in size . the wide upper portion of the mass has 2 small appendages at either corner that are each 1 cm long ( fig . the narrow lower end of the mass was firm in consistency ( arrow in fig . 2 ) . we opened the mass and observed amniotic fluid and two dead fetuses , each 6 cm in length ( fig . the mass wall was thin in the upper segment and thick in the lower segment . the portion of the mass representing the cervix was solid and did not have an internal opening or cervical canal , as confirmed using a fistula probe . tissue samples taken from the upper and lower portions of the specimen for histopathological analysis both exhibited the smooth muscle fiber arrangement of uterine tissue . one year later , a hysterosalpingogram showed a normal uterine cavity and two patent fallopian tubes ( fig . a few months later , she became pregnant , and she had a full term pregnancy with normal vaginal delivery . she had four subsequent pregnancies over 9 years that all reached full term and all resulted in normal vaginal deliveries . there was a lack of a full medical history and poor communication between the gynecologist and the ultrasonographist . thus , despite the proficiency of the ultrasonographist , during two examinations , he did not thoroughly examine all pelvic organs once he detected a twin miscarriage in what appeared to be a uterine cavity . if he had examined all pelvic organs , he should have detected the empty normal uterus just beside the gravid abnormal uterus as it is clear in the post - operative hystersalpingography ( fig . hysteroscopy could have been useful in this case , but unfortunately , the only available hysteroscope in the hospital was broken - down . most of the congenital anomalies of the uterus are due to a fault in one or more step in the fusion process of the mullerian ducts . in this case , however , the accessory uterus most probably developed as a growth from the right mullerian duct generating an incompletely developed separate accessory uterus ( non - communicating , cavitated accessory uterus ) acum . most probably , the ovum entered the accessory uterus through the small appendages on the sides of the dome , which represent rudimentary fallopian tubes . the ovum likely became fertilized by sperms passing through the normal cervical canal of the normal uterus , uterine cavity , fallopian tube , and the peritoneal cavity , or the sperms may have travelled through the lymphatic vessels of the accessory uterus to fertilize the ovum inside it . 1.a deficient case history , lack of communication between the clinicians and the ultrasonographist and repeated shifting of the patient from one doctor to another , all led to the misdiagnosis and mismanagement of this patient.2.conception can occur in acum , however , it is very rare and may be difficult to diagnose . a deficient case history , lack of communication between the clinicians and the ultrasonographist and repeated shifting of the patient from one doctor to another , all led to the misdiagnosis and mismanagement of this patient . conception can occur in acum , however , it is very rare and may be difficult to diagnose . harith m. alkhateeb : surgery for the patient , writing of original text , communication with the editorial team . enas m. yaseen : the last gynecologist dealing with the patient , periodical clinical examinations and follow up of the patient . a copy of the written consents are available for review by the editor - in - chief of this journal .

introduction : desmoid tumor of bone is a rare benign tumor . it is reported commonly in mandibular and meta - diaphyseal region of long bones . we report involvement of unusual site in ilio - acetabular region with breach in articular cartilage.case report : a 40 year old female presented with pain in the left hip . radiologically , a lytic lesion at ilio - acetabular region was seen . intra - operatively breach in acetabular roof was seen which was missed in the scan . curettage and defect reconstruction was done . histopathology reported as desmoid tumor . 20 months post - operatively patient was symptom free.conclusion:desmoid tumor is a rare bone tumor . this case report emphasizes about the rarity of the lesion in this location and the rare chances of breach in articular cartilage of the joint . the chances of recurrences are high with intralesional curettage . highlightsobtaining a detailed medical history is important , and we should not depend on examination tools when dealing with patients.unexplained findings should raise suspicions regarding other possible diagnoses , and the same non - conclusive procedures should not be repeated.following this advice can prevent unnecessary operative procedures and the complication of perforation of the uterus .

a 19-year - old female with no significant past medical history or medication use presented to our hospital in april 2010 with severe nephrotic syndrome characterized by marked fluid retention , heavy proteinuria ( protein : creatinine ratio of 1244 mg / mmol ) , hypoalbuminaemia ( 13 g / l ) and elevated total serum cholesterol ( 9.3 mmol / l ) . renal function was preserved with a serum creatinine of 42 mol / l , and there was no haematuria evident on urine microscopy . lupus and vasculitis markers , hepatitis serology and serum electrophoresis were negative . a renal biopsy was performed which revealed normal glomeruli , tubules and interstitium . the patient was commenced on prednisolone 50 mg daily ( 1 mg / kg ) in addition to ramipril 5 mg daily , simvastatin 10 mg daily and warfarin therapy . complete remission was achieved within 2 months and prednisolone dose was reduced to 25 mg daily . soon after , and against medical advice , the patient abruptly ceased her prednisolone and within 72 h had a relapse of disease with recurrence of gross peripheral oedema , hypoalbuminaemia of 13 g / l ) and proteinuria of 1030 mg / mmol ( from 0.06 mg / mmol ) . despite recommencing high - dose steroids ( 60 mg / day ) , the disease thereafter remained refractory to steroid therapy with persistent gross oedema , nephrotic - range proteinuria and hypoalbuminaemia ( albumin of 9 g / l ) . after failing to respond to 3 months of high - dose steroids , the patient was commenced on cyclophosphamide . in light of the patient 's history of non - compliance and in an effort to reduce drug exposure and toxicity , pulse intravenous cyclophosphamide was administered in incremental doses of 0.5 , 0.75 and 1.0 g / m 1 month apart . during this period two months after the third pulse of cyclophosphamide was administered , there was no evidence of disease response with ongoing gross oedema , hypoalbuminaemia ( albumin 14 g / l ) and heavy proteinuria ( 1476 mg / mmol ) . at this point , it was decided to trial the monoclonal anti - cd20 antibody rituximab ( rtx ) , with two doses of 500 mg given 2 weeks apart . complete remission was rapidly induced within 1 month of rtx therapy with resolution of oedema , rising albumin to 31 g / l and absent proteinuria ( figure 1 ) . 1.changes in serum albumin ( g / l ) and urine protein : creatinine ratio ( mg / mmol ) over time , relative to treatment modality . changes in serum albumin ( g / l ) and urine protein : creatinine ratio ( mg / mmol ) over time , relative to treatment modality . the patient has remained in complete remission for more than 32 months since the two doses of rtx were administered in february 2011 . this is despite recovery of cd19 counts back to normal levels 24 months after treatment . mcd is the most common cause of nephrotic syndrome in children and accounts for 1025% of cases in adults . close to 75% of cases will respond initially to a course of high - dose glucocorticosteroids but relapse commonly occurs . steroid - sparing agents like calcineurin inhibitors and cyclophosphamide are often used for frequently relapsing or steroid - dependent mcd . besides the significant adverse side effects of these agents , a large proportion of patients relapse after cessation of these medications . while there was some suggestion that oral cyclophosphamide may be more successful than iv cyclophosphamide in achieving disease remission in refractory mcd , larger clinical trials have suggested that both forms of therapy are equivocal with respect to relapse rates and toxicity . there are an increasing number of reports of success of rtx therapy for steroid- and cyclosporine ( csa)-dependent nephrotic syndrome in paediatric populations [ 4 , 5 ] . a single rtx infusion also appeared to improve response to csa therapy and had significant steroid - sparing effects in children with steroid-dependent nephrotic syndrome ( sdns ) who had developed secondary resistance to csa . in a long - term follow - up study of 37 children who received rtx for sdns , 26 patients remained in remission after 12 months and 7 remained in remission after 24 months without further maintenance immunosuppression . however , response to rtx has been mixed in cases of steroid-resistant nephrotic syndrome ( srns ) in children . reported the successful use of rituximab in five patients with srns , two of which had mcd . . carried out a multi - centre questionnaire - based study in which the response rate in children with srns was as high as 44% . in another case series of four children with srns , including one case of mcd , all children failed to achieve sustained remission after a single dose of rituximab , despite complete b - cell depletion . in adults , the first case report of the efficacy of rtx in multi - relapsing mcd was published in 2007 . since then there have been a number of reports of success with rituximab in steroid - dependent mcd [ 12 , 13 ] . munyentwali et al . analysed the outcome of 17 adult patients who were treated with rtx for steroid - dependent mcd over a mean follow - up period of 29.5 months . after the first course of rtx , 11 patients ( 65% ) did not relapse after a mean follow - up of 26.7 months . twelve of the 17 patients ( 70% ) were free of any immunosuppressant drugs at last follow - up . there are minimal data available regarding the use of rtx in steroid - resistant mcd in adults . the first case report , published in 2008 , describes an adult patient with steroid- and mycophenolate mofetil - resistant mcd who remained in complete remission 1 year after initiation of rtx therapy . a further case report was of a 23-year - old patient with steroid- and csa - resistant mcd treated with two doses of rxt with sustained complete remission at the end of 1 year . the second dose of rtx was given at 6 months in response to a recovery of b - cell counts . a long - term follow - up study was published recently by bruchfeld et al . on 16 adult patients with biopsy - proven mcd of which 13 were steroid dependent , 2 multi - relapsing and 1 steroid and multi - drug resistant . in this study , the rtx dose varied from 1000 to 2800 mg . complete remission was achieved after initial rtx treatment in 13 patients . in the remaining cases , partial remission was achieved in all except the patient with steroid- and multi - drug - resistant disease . relapses can often occur after the reappearance of cd19 cells , although this is not always the case [ 12 , 14 ] . guigonis et al . found a consistent association between relapses ( 3 of 22 paediatric patients ) and an increase in cd19 cells which occurred at a median time of 11 months ( range 839 months ) . sellier - leclerc et al . evaluated disease outcome after a minimum cd19 depletion period of 15 months obtained by repeated rtx infusion and found that it induced long - term remission even after definitive cd19 recovery in almost two thirds of the paediatric patients without the use of maintenance oral immunosuppressive drugs . the rtx dosing has varied greatly in various reports including a single dose , doses of 500 or 1000 mg given at one or two time - points and 375 mg / m once weekly for 4 weeks . in the study by munyentwali et al . we report a unique case of steroid- and cyclophosphamide - resistant mcd responding rapidly to two doses of rtx at 500 mg each , with sustained complete remission even after cd19 recovery . as discussed , rtx has been shown to be efficacious in a growing number of small case series in steroid - sensitive and steroid - dependent mcd ; however , reported success of its use in multi - drug - resistant disease , particularly in adults , remains limited . in our case , clearly , a well - conducted multi - centred clinical trial of rtx for mcd is warranted either as a first - line agent or in cases of steroid - dependent and/or -resistant disease . the results presented in this paper have not been published previously in whole or part , except in abstract format . among all emergency cases the ratio of incarcerated / strangulated hernias differs across the countries . but it is well known that hernia is the common cause of small bowel obstruction , and mortality and morbidity rates increase in case of strangulation . during acute ischemic conditions , metal binding capacity of albumin this change is quantifiable and commonly known as ischemia modified albumin ( i m a ) . recently , i m a measurement has been proposed as a sensitive marker for the diagnosis of myocardial ischemia presenting with typical acute chest pain . however , i m a levels are also known to increase in other ischemic conditions , as well as to be an indicator of oxidative stress , and may not be specific for cardiac ischemia . recently , turedi et al . showed that the level of i m a may be of use in the diagnosis of pulmonary embolism , another ischemic condition . incarceration is the state of an external hernia which can not be reduced into the abdomen . roughly 5% of all patients having an operation for external hernia are explored as an emergency . strangulated external hernia is a relatively common and serious surgical emergency and accounts for most of the deaths from this condition . clinical validation of any test for ischemia is difficult as there is no accepted diagnostic gold standard . and the preoperative diagnosis or prediction of ischemia is important for surgeons because ischemia changes the operative strategy . thus we planned this study and aimed to show relation between the intestinal ischemia and i m a . our study was initiated after anakkale onsekiz mart university ( comu ) animal ethics committee approval ( approval number 2010/11.02 ) and took place in comu laboratories . twenty wistar albino rats were used weighing 153207 g and were housed in optimum conditions . in this study rats , they were kept for two weeks in cages with five animals each and 12 : 12-hour artificial light to dark cycles . we aimed to mimic incarceration by short clamping ( 45 minutes ) and strangulation by 6 hours clamping . ketamine hcl ( ketalar , pfizer , turkey ) 35 mg / kg and xylazine ( basilazin flacon % 2 , turkey ) 5 mg / kg were administered intraperitoneally as anesthetic agents . after midline laparotomy , mesenteric vascular structures of small intestine were clamped by a vascular clamp . then blood samples were taken [ for i m a ( ima1 ) and lactate dehydrogenase ( ldh ) ] and midline incision was closed separately by polypropylene sutures . after midline laparotomy , mesenteric vascular structures of small intestine were ligated by 2/0 polypropylene sutures and midline incision was closed separately by polypropylene sutures . midline laparotomy was done again at 6th hour , intestinal necrosis were seen , and blood samples were taken [ for i m a ( ima2 ) and ldh ] . after midline laparotomy then blood samples were taken [ for i m a ( ima1 ) and ldh ] and midline incision was closed separately by polypropylene sutures . but incarceration is a known ischemia reperfusion process and total vascular clamping is a widely used method to investigate ischemia reperfusion in rats . so we used this method to simulate incarceration in group i. also total occlusion of all vascular support of the small intestine is used in group ii to provide the smaller amounts of ischemic / necrotic tissue in group i and group ii and to prevent the potential effect of ischemic / necrotic tissue amount on i m a levels . reduced cobalt to albumin binding capacity ( i m a levels ) was analyzed using the rapid and colorimetric method developed by bar et al . . results higher than 0,4 absu were estimated as ischemia and lower than 0,4 absu were estimated as normal . kruskal - wallis test was used to compare the groups and probability ( p ) values less than 0.05 were considered significant . the significance of the parameters in kruskal - wallis test was confirmed by mann - whitney u test and p values less than 0,016 were considered significant . kruskal - wallis test is the standard variance analysis test to compare the means of 3 or more groups . kruskal - wallis test gives us the difference between the groups , but it does not give any idea about which group is the cause of this difference . we used mann - whitney u test with bonferroni correction ( the p value for mann - whitney u test by bonferroni correction is found to be 0,05/number of groups = 0,016 for this study ) . mean ima1 levels of group i , group ii , and group iii were 0,41 0,01 ( range 0,400,45 ) , 0,46 0,16 ( range 0,440,48 ) , and 0,34 0,03 ( range 0,280,39 ) , respectively mean ldh levels were 819,71 34,53 ( range 766874 ) , 808,57 24,69 ( range 774841 ) , and 514,14 19,92 ( range 487545 ) for groups i , ii , and iii , respectively . the difference of i m a levels between group i and group ii was not significant at 6th hour ( ima2 ) ( p : 0,71 ) during acute myocardial ischemia , the ability of binding ions such as copper , zinc , and cobalt is decreased ; therefore a form of albumin is produced , which is described as ischemia modified albumin ( i m a ) . the i m a was registered by the united states food and drug administration as a marker of myocardial ischemia . the test is based on the decreased ability of albumin to bind cobalt due to the structural change of nh2 which develops in the ischemic environment . it is important to emphasise that the decreased ability of albumins to bind cobalt occurs in hypoxia , acidosis , sodium and calcium pump malfunctions , and tissue damage caused by free radicals [ 7 , 8 ] . it has been shown that the concentration of i m a increases within a couple of minutes from the onset of ischemia , remains elevated until 6 to 12 hours , and returns to its normal range after 24 hours . but recent studies showed that i m a increases in mesenteric ischemia , stroke , and cerebrovascular accidents as well [ 1 , 10 ] . found significantly higher i m a levels in superior mesenteric artery thromboembolism and they concluded that i m a can be used in the diagnose of mesenteric ischemia . patients requiring emergency repair tend to be a member of an elderly and unfit population [ 1315 ] . ge et al . concluded that the main causes of high incarceration rates are economic problems and lack of insurance . morbidity and mortality risks of incarcerated hernias are increased by longer duration of symptoms [ 1315 , 19 , 20 ] , type of hernia ( femoral rather than inguinal hernia ) , a high american society of anesthesiology ( asa ) score [ 14 , 17 , 19 , 21 ] , the presence of comorbidities [ 14 , 15 ] , and the presence of bowel necrosis [ 14 , 17 , 19 ] . this study aimed to predict necrosis by i m a levels and explore the impact of ldh . we can evaluate asa score , duration of symptoms , and the presence of comorbidities preoperatively , but the need for an intestinal resection is unclear until surgery . this has been reported to be the case even for experienced surgeons where the sensitivity has been reported to be , at best , 48% . maeda et al . reported that the serum creatine phosphokinase ( cpk ) level was a useful marker for the diagnosis of a strangulated hernia , and ohira et al . found that cpk , lactate dehydrogenase , and metabolic acidosis , in addition to ultrasound and enhanced computed tomography , were of value in diagnosing strangulation of the intestine in some cases . a definitive diagnosis of strangulation of the intestine , however , can only be made during a surgical exploration [ 5 , 25 ] . the current series showed a significant increase in the need for a resection of the small intestine when the time from onset of symptoms to surgery was more than 12 hours ; the rate of resection of the small intestine in patients presenting within 12 hours was 14.3% , but it increased to 50% in patients presenting after 12 hours or more ( p = 0.0392 ) . reported that the resection rate in the bowel operated on within 24 hours was 7% , but when the time increased to 48 hours or more , the resection rate increased up to 27% . 's results.ldh was elevated in both incarceration and strangulation groups but it was failed to predict necrosis . in this study we aimed to predict intestinal necrosis by i it is important to predict necrosis , because incarcerated hernias are more frequently seen in elderly patients , and their prevalence in the over-60-year - old population ( added comorbidities and anesthetic risks ) has been reported to be 9.8% compared with 1.8% for younger patients . besides it is known that general and spinal anesthesia are both associated with higher rates of serious postoperative complications . the prediction of necrosis can give both the surgeon and the patient the chance of local anesthesia . unfortunately the disadvantage of this study is the lack of evaluating other predictive values like cpk levels and since this study is an animal study , pospective human studies must be performed using i m a to be able to suggest that i m a is a gold standard to predict strangulation in the hernia sac . i m a is a new marker of ischemia and its place in general surgery is not well known yet . further studies should be done to predict intestinal necrosis in the hernia sac , to be able to protect old and unfit patients from the anesthesia associated mortality and morbidities .

we report a case of steroid- and cyclophosphamide - resistant nephrotic syndrome secondary to minimal - change disease occurring in an otherwise healthy 19-year - old female , responding rapidly to two doses of rituximab therapy . complete disease remission has been sustained up to last follow - up ( 32 months ) despite cd19 recovery . literature review suggests emerging evidence that rituximab may have a role to play in recurrent and/or refractory minimal - change disease . purpose . to evaluate the predictive effect of i m a in incarcerated hernias . methods . three groups ( n = 7 ) of rats were operated . group i aimed to mimic incarceration , group ii aimed the strangulation , and group iii was the sham group . i m a and ldh measurements were made . results . i m a levels were significantly higher in strangulation mimicking group and i m a levels were normal at postoperative 6th hour in incarceration mimicking group . ldh levels were significantly higher in both incarceration and strangulation mimicking groups . conclusion . i m a seems to be an effective marker in incarcerated hernias to predict necrosis . but we need further studies to generalise this hypothesis .

iran is in the thalassemic belt with a frequency of about 6 -10% in the northern and southern provinces ( 1 ) . cancer antigen 15.3 ( ca15.3 ) , a product of muc1 gene ; is an epithelial mucin and its high level is associated with breast cancer . in patients with breast cancer , evaluation of ca15.3 has not been recommended by national comprehensive cancer network guidelines for surveillance purposes , but its monitoring suggested to detect recurrences of cancer evaluating therapeutic response of advanced disease and the assessment of prognosis in patients with early breast cancer ( 2,3 ) . also it may be high in nonmalignant conditions such as inflammatory processes and some hematologic disorders like sickle cell anemia and thalassemia major ( 4).improving management of thalassemia major has led to increased life expectancy and subsequently increased frequency of age related conditions such as malignancies ( 5 - 7 ) . as a result , some investigators have opted to check serum tumor markers in these patients . however , they showed an increased frequency of serum ca15.3 independent from the presence of malignancy in these patients . the suggestive mechanism is erythroid hyperplasia which could result in high level of muc1 expression on bone marrow - appoptosing progenitor cells ( 5 , 8) . up to our knowledge , evaluation of serum ca15.3 in patients with thalassemia minor has not been conducted . our hypothesis was that patients with beta - thalassemia minor has a higher serum level of ca15.3 compared to healthy individuals and patients with thalassemia major but with a milder degree . present study was designed to measure the serum level of ca15.3 in patients with beta - thalassemia minor compared to healthy individuals and to patients with cancer . also we aimed to determine the frequency of beta - thalassemia minor in a population of young patients with malignancies . in this cross - sectional study , 32 carriers of beta - thalassemia , 25 healthy individuals , and 49 patients with cancer were recruited from february to december 2011 . carriers of beta - thalassemia were non - randomly selected from the volunteers referred for premarital screening in an outpatient clinic affiliated to shiraz university of medical sciences , southern iran . all patients with cancer who referred to pediatric hematology - oncology clinic of shiraz university of medical sciences in the study period were recruited . healthy controls were randomly selected from healthy individuals who referred to pediatric hematology - oncology clinic for checkup examination . patients with cancer were subdivided to 2 groups of with and without beta - thalassemia minor . in all participants , the ca 15.3 was checked by canag ca 15 - 3 eia kit , sweden in all participants . the protocol for this study was approved by the ethics committee of shiraz university of medical sciences . written informed consent was obtained from all participants or their parents . test of normality was done by kolmogorov - smirnov test ( ca15.3 , p=0.2 and age , p=0.156 ) . comparison of serum level of ca15.3 level and age among various groups were done by anova test . to find the statistically significant difference among different groups , post hoc tukey hsd test was performed . sex distribution was compared among the studied groups using chi - square test , and p value less than 0.05 was considered statistically significant . participants were divided to 4 groups including : group 1 , beta - thalassemia minor ; group 2 : patients with cancer and without beta - thalassemia minor ; group 3 : patients with cancer and beta - thalassemia minor , and group4 : healthy individuals . comparisons of the mean age and sex ratio as well as mean serum ca 15.3 level among the four groups of participants are shown in table 1 . * statistically significant , ca ag 15 - 3 , cancer antigen 15.3 carriers of beta - thalassemia were significantly older than the other groups ( p<0.0001 for comparison of this group with each of the other three groups based on post hoc tukey hsd test ) . male to female ratio was not significantly different among the studied groups ( p=0.804 ) . the serum levels of ca 15.3 in all participants were in the normal range ( < 35 u / ml ) and its concentrations shown no significant difference among various groups of the participants ( p=0.723 ) . age was not significantly correlated with the serum level of ca 15.3 ( r= 0.039 , p=0.702 ) . as shown in table 2 , of 49 patients with cancer , 96% had hematologic malignancies with the highest frequency for acute lymphoblastic leukemia ( 37 patients ) . acute lymphoblastic leukemia : ( all ) , acute myeloblastic leukemia : ( aml ) , hodgkin lymphoma : ( hl ) , non hodgkin lymphoma : ( nhl ) of 49 patients with malignancies 11 ( 22.4% ) diagnosed as thalssemia minor by their cbc and hemoglobin electrophoresis results . we evaluated serum level of ca 15.3 in carriers of beta - thalassemia by comparing them to the three other groups including patients with cancer and those with or without beta - thalassemia minor as well as healthy individuals . we did not find any significant difference with regard to the ca 15.3 concentrations among the groups . moreover , we expected that the higher serum level of ca 15.3 in patients with cancer compared to the reference range . this is in contrast with the results of other reports which showed increased levels of ca 15.3 among patients with solid tumors ( 9 ) . we could not find any publication that has investigated the serum levels of ca 15.3 in patients with hematologic malignancies . as we mentioned most of our patients had hematologic malignancies and mostly acute lymphoblastic leukemia . this result suggests that the ca 15.3 may not be associated with hematologic malignancies and thus it can not be used as a tumor marker . we found a frequency of 22.4% ( 11 out of 49 ) of beta - thalassemia minor in the group of patients with malignancies . compared to the prevalence rate of about 10% in the general population therefore the suggestive mechanism could be shorter life span of red blood cells , and theoretically erythroid hyperplasia secondary to ineffective erythropoiesis which could cause errors in cell division leading to hematologic malignancies . these findings did not support the results of the previous study in our center which showed protective effect of minor thalassemia against childhood malignancies ( 1 ) . our study was limited due to lack of matched matched of carriers for beta - thalassemia with other groups because most of participants in this group selected from persons who were screened for marriage . but other groups were selected from younger children and persons referred to pediatric hematology - oncology clinic . however , we did not find a significant correlation between age and serum ca 15.3 concentration in our participant . moreover our research was a cross - sectional study and we can not conclude strongly about the relationship between cancer and beta - thalassemia minor . to document the accurate correlation we need a cohort study for this matter . moreover , during the course of our study , most of our patients in the cancer group had hematologic malignancies . the values of ca 15.3 serum levels were in the normal range for both in carriers of beta - thalassemia and in patients with malignancies . no correlation was found between ca 15.3 serum level with thalssemia minor or with childhood malignancies . compared to normal population , a high proportion ( 22.4% ) of thalssemia minor was observed in patients with cancer in our study . future prospective studies are needed to evaluate the relationship between cancer and beta - thalassemia minor accurately . the accelerated emergence of antibiotic resistance among the prevalent pathogens is of global health concern . enterococcus , is one such pathogen which is the leading cause of nosocomial bacteremia , urinary tract infections ( uti ) , and surgical site infections . enterococcus resistance to antimicrobial agents to which the genus streptococcus are generally susceptible and its ability to transfer the drug resistance genes from vancomycin - resistant strains to staphylococcus aureus is of concern . the therapeutic challenge of multiple - drug resistant ( mdr ) enterococci , identifies them as important nosocomial pathogens . enterococci infections have traditionally been treated with cell wall inhibitor agents in combination with an aminoglycoside . reduced susceptibility to -lactam antibiotics and vancomycin ; in combination with a high level aminoglycoside resistance ( hlar ) interferes with the penetration of the aminoglycoside into the bacterial cytoplasm , thus making the antibiotic synergism ineffective . hence , this study was designed to identify the magnitude of enterococcal infections and their antibiotic susceptibility pattern in a tertiary care hospital in the eastern region of india . following approval from our institutional ethics committee , clinical samples were collected over a period of 1-year ( february 2011 to january 2012 ) at a tertiary care hospital in kolkata , india . the study population included patients of all age groups and both sexes with suspected nosocomial infections ( infection developed after 48 h of hospital stay ) . patients with infections at the time of admission , within 48 h of hospital stay or 30 days after discharge were excluded . the clinical samples of urine , wound swab / pus , high vaginal swab and blood were inoculated on blood agar and macconkey agar . the causative bacteria were identified on the basis of colony characteristics , gram stain morphology , motility and biochemical tests ( catalase test , growth on bile aesculin agar , tolerance to 6.5% nacl , arginine dihydrolase test , and fermentation of arabinose , mannitol , raffinose , and sorbitol ) . the isolates were subjected to antimicrobial susceptibility testing by kirby - bauer disk diffusion method , as per clsi recommendations , using commercially available 6 mm disks ( himedia , mumbai , india ) on mueller - hinton agar . the disks used were vancomycin ( 30 g ) , ampicillin ( 10 g ) , erythromycin ( 15 g ) , ciprofloxacin ( 5 g ) , linezolid ( 30 g ) . for high - level gentamicin resistance ( hlgr ) detection , gentamicin ( 120 g ) disc was used . the diameter of the zone of inhibition of each antibiotic was measured and interpreted as sensitive , intermediate sensitive or resistant according to clsi criteria . for hlgr , resistance was indicated by no zone , and susceptibility , by a zone of diameter 10 mm . enterococcus faecalis atcc 29212 and e. faecalis atcc 51299 were used as the susceptible and resistant quality control strains . brain - heart infusion agar ( hi media , mumbai ) was supplemented with different concentrations of vancomycin . the test organism was grown in broth and the turbidity matched with mcfarland 0.5 standard ( approximately 1.5 10 cfu / ml ) . the isolates were subjected to antimicrobial susceptibility testing by kirby - bauer disk diffusion method , as per clsi recommendations , using commercially available 6 mm disks ( himedia , mumbai , india ) on mueller - hinton agar . the disks used were vancomycin ( 30 g ) , ampicillin ( 10 g ) , erythromycin ( 15 g ) , ciprofloxacin ( 5 g ) , linezolid ( 30 g ) . for high - level gentamicin resistance ( hlgr ) detection , the diameter of the zone of inhibition of each antibiotic was measured and interpreted as sensitive , intermediate sensitive or resistant according to clsi criteria . for hlgr , resistance was indicated by no zone , and susceptibility , by a zone of diameter 10 mm . enterococcus faecalis atcc 29212 and e. faecalis atcc 51299 were used as the susceptible and resistant quality control strains . brain - heart infusion agar ( hi media , mumbai ) was supplemented with different concentrations of vancomycin . the test organism was grown in broth and the turbidity matched with mcfarland 0.5 standard ( approximately 1.5 10 cfu / ml ) . 101 of 1194 urine isolates , 30 of 662 wound swab / pus isolates , 13 of 307 blood isolates and 09 of 203 high vaginal swab isolates were identified as enterococci [ table 1 ] . all isolates were further speciated as e. faecalis ( 90.85% ) , enterococcus faecium ( 8.50% ) and enterococcus gallinarum ( 0.65% ) . most urinary isolates were from maternity ward ( n = 25 ) , followed by gynecology ( n = 23 ) , and pediatric ward ( n = 10 ) respectively . enterococcal wound infections were also most commonly reported from gynecology ( n = 7 ) followed by surgical wards ( n = 5 ) . enterococci were the causative pathogen in high vaginal swab in patients from maternity , gynaecology and itu . mic of vancomycin observed in all enterococcal isolates was < 1 g / ml which corroborates the disc diffusion test result . the spectrum of disease produced by enterococci varied from uti , wound infection , soft tissue infection to bacteremia . urinary tract instrumentation or catheterization , genitourinary pathology , prior use of antibiotics , prolonged hospitalization were some of the predisposing factors for enterococcal infections . urinary tract was the most common site of enterococcal infection ( 66.01% ) in this study which often occurred in catheterized patients . the next common infection was wound infection ( 19.60% ) followed by bacteremia ( 8.50% ) which corroborates with the studies from different regions of india [ figure 1 ] . in india , e. faecalis is the predominant enterococcal species , which accounts for 80 - 90% of all clinical isolates , which is followed by e. faecium ( 5 - 15% ) . however , a progressive increase in e. faecium infections has been reported and is found to be more resistant to penicillin and aminoglycosides , which is attributed to production of the enzyme , 6-acetyl transferase and more penicillin - binding proteins . in our study , e. faecalis was the commonly isolated species followed by e. faecium and e.gallinarum , the incidence rates were comparable with previous reports . comparison between isolation rates of enterococcus in different clinical samples from different regions of india all enterococci isolates were found to be sensitive to vancomycin and linezolid which was consistent with other studies from india , however the prevalence of vancomycin resistant enterococci ( vre ) in india is reported to be between 0% and 30% . almost half of the isolates were resistant to ciprofloxacin ( 49.67% ) and gentamicin ( 45.75% ) [ figure 2 ] . in contrast , ampicillin showed a high level of sensitivity ( 77.12% ) among the nosocomial isolates [ table 2 ] . resistance to ciprofloxacin was also less prevalent among the wound isolates in comparison with the urine isolates . ampicillin and gentamicin showed a higher level of resistance among the wound isolates compared with urine isolates . isolates from nosocomial bacteremia showed a higher level of sensitivity to each of the drug tested [ table 3 ] . antibiotic resistance pattern of enterococcus as evident in different studies antimicrobial sensitivity pattern of the isolates in nosocomial infections comparison of percentage prevalence of antibiotic resistance among isolates from different sources the hlar to gentamicin was observed in 45.75% isolates with 43.88% in e. faecalis and 69.23% in e. faecium [ table 4 ] . hlar to gentamicin is universally reported to be in the range of 1 - 48% ( mean , 22.6 12.3 ) , although with an increasing trend recently . hlar to gentamicin nullifies the efficacy of combination therapy , which is used to treat serious enterococcal infections . nevertheless , empirically chosen combination therapy with ampicillin and gentamicin would be effective in 54.25% of nosocomial infections [ figure 3 ] . interestingly 7% of the isolates ( n = 11 ) in our study were mdr and vancomycin or linezolid were the only available option for treating these patients . the mdr strains were mostly reported from gynecology ( n = 3 ) and maternity wards ( n = 2 ) . comparison between -lactam ( ampicillin ) and aminoglycoside ( gentamicin ) susceptibility patterns of enterococci isolates from different sources ( n = 153 ) ( am : ampicillin , g : gentamicin , s : sensitive , r : resistant ) our study reveals the prevalence of high degree of resistance to macrolide and fluoroquinolone among the nosocomial enterococcal isolates , thereby limiting the use of these drugs for therapeutic purposes . the resistogram of the enterococcal isolates varied among specimens from different wards , but the pattern was constant among isolates within a particular ward . hence , the nosocomial outbreak in our hospital had not been disseminated from a single strain though isolates from a particular ward might be epidemiologically linked . the present study also revealed that despite recent trends of increasing resistance to aminoglycosides , a combination therapy of -lactam and aminoglycoside as first - line drugs would be currently the best choice . vancomycin or linezolid therapy should be restricted for use in patients infected with mdr strains only . judicious use of vancomycin and linezolid in serious infections and appropriate infection control measures would probably recede the possible emergence of vre outbreaks in our geographical area .

background : high serum level of cancer antigen 15.3 ( ca15.3 ) has been reported in some malignant and nonmalignant conditions including thalassemia major which could have been resulted from ineffective erythropoiesis . we aimed to evaluate the serum level of ca15.3 in carriers of beta - thalassemia by comparing them with cancer patients and healthy individuals . methods : this cross - sectional study was done from february to december 2011 in southern iran . participants consisted of 32 subjects with beta - thalassemia minor , 49 with cancer and 25 healthy individuals . the serum levels of ca15.3 were measured and compared in different groups . results : the serum levels of ca 15.3 in all participants were in the normal range ( < 35 u / ml ) . also it did not significantly differ among various groups of the participants ( p=0.723 ) . age was not significantly correlated with the serum level of ca 15.3 ( r= 0.039 , p=0.702 ) . the most frequent cancer in the group of patients with malignancies was hematologic malignancies ( 96% ) with the highest frequency for acute lymphoblastic leukemia ( 37 patients ) . frequency of thalassemia minor in patients with cancer was 11 ( 22.4% ) . conclusion : no correlation was found between ca 15.3 serum level with beta - thalssemia minor or with childhood malignancies . compared to general population , a high proportion of beta - thalssemia minor was observed in patients with cancer in our study . future prospective studies are needed to evaluate the relationship between cancer and beta - thalassemia minor accurately . background : resistance to commonly used antibiotics by enterococci causing nosocomial infections is of concern , which necessitates judicious , responsible and evidence - based use of antibiotics . the present study was conducted to review the prevalence and identify therapeutic options for nosocomial enterococcal infections in our tertiary care hospital.materials and methods : isolates identified by morphological and biochemical characteristics were tested for antibiotic susceptibility using kirby - bauer method.result:153 of 2096 culture positive clinical samples comprised of 101 urine , 30 wound swab / pus , 13 blood and 09 high vaginal swab isolates were identified as enterococcus faecalis ( 90.85% ) , enterococcus faecium ( 8.50% ) and enterococcus gallinarum ( 0.65% ) . enterococci accounted for 8.45% , 4.53% , 4.23% , 4.43% of urinary , wound swab or pus , blood , high vaginal swab isolates respectively , causing 7.3% of all nosocomial infections . significant number of enterococci isolated from nosocomial urinary tract infection ( 66.01% ) and wound infections ( 19.6% ) were multidrug resistant ( mdr ) . although all isolates were sensitive to vancomycin and linezolid , resistance to erythromycin ( 71.24% ) and ciprofloxacin ( 49.67% ) was frequently observed . high - level gentamicin resistance was observed in 43.88% , and 61.53% of e. faecalis and e. faecium isolates respectively . minimal inhibitory concentration of vancomycin of all the isolates were 1 g / ml . 7% of the enterococcal isolates were mdr strains and vancomycin or linezolid were the only effective antibiotics.conclusion:a combination of vancomycin and/or linezolid were effective against enterococci causing nosocomial infections in our tertiary care facility , nevertheless continuous and frequent surveillance for resistance patterns are necessary for judicious and evidence based use of antibiotics .

the wideness of interest of a scientific discipline , and its impact on knowledge and the research progress may be estimated through the number of journals issued in its field , the quantity of articles published in a given timespan , and the number of citations these articles obtained . no doubt , this is a rough estimate but it may allow to quantitate and compare the data taken from qualified databases , and to foresee future developments . during the last fifteen years , the number of journals published in the biomedical field has significantly increased : for instance , based on the thomson reuters journal citation report ( https://jcr.incites.thomsonreuters.com ) the journals in the subject categories of biology increased from 51 to 85 , those in cell biology from 147 to 184 , in medicine ( experimental & research ) from 74 to 123 , and in oncology from 103 to 211 , with an impressive ( though hardly computable ) surge of published articles . research in biomedicine has taken advantage of the progress in several disciplines , first of all in molecular biology and related omics . microscopy and histochemistry also played a major role , especially in recent years , by providing new instrumentations and refined techniques . in the attempt to assess the impact of histochemistry in the biomedical field , it may be interesting to look at the articles recently published in qualified journals , reporting the results of investigations performed by histochemical methods . browsing the records of pubmed ( http://www.ncbi.nlm.nih.gov/pubmed/ ) , web of science ( http://thomsonreuters.com/en/products-services/scholarly-scientific-research/scholarly-search-and-discovery/web-of-science.html ) and scopus ( http://www.scopus.com/ ) , the articles published in qualified journals where histochemistry or immunohistochemistry are present in the title / abstract or as key - words were about 87,000 , 130,000 and 132,000 respectively , in the last five years ( the numerical differences are essentially due to the different journals which are indexed : more medicine - oriented in pubmed , and more widely biology & medicine - oriented in web of science and scopus ) . by grouping these publications in subject categories , according to their contents ( figure 1 ) , it is apparent that more than 40% deal with medical subjects ( pathology & experimental medicine ) ; articles on differentiation & stem cells or neurons & muscle ( more than 15% and 10% , respectively ) , as well as those on methods ( about 10% ) are also present at high percentages . about 5% are those on animal tissue biology and hard tissue & matrix , and only few on tissue engineering or on plant tissue biology . thus , in these five recent years , biomedical studies where histochemistry has been used as a major experimental approach included a wide variety of subjects , from basic to applied research on both humans and other animal or plant organisms . among the purely histochemical journals , the european journal of histochemistry - ejh has traditionally been devoted to publish papers on functional cell and tissue biology in animals and plants , cell differentiation and development , cell - to - cell interaction and molecular trafficking , nerve and muscle cell biology , with special attention to the cellular basis of diseases . it is worth observing ( figure 1 ) that some differences exist in the distribution of subjects of the papers published in the european journal of histochemistry during the same five - years period , compared to the overall histochemical articles in the literature . consistent with the latter ones , the majority ( about 35% ) of the published articles were on pathology & experimental medicine , and the percentages found for hard tissues & matrix , neurons & muscle and plant tissue biology were also similar as those for the whole histochemical production . on the contrary , the percentage of papers published on animal tissue biology , on methods and on tissue engineering was higher in the european journal of histochemistry , whereas the one on differentiation & stem cells was lower . looking at the distribution of articles on these subjects during the last five years ( figure 2 ) may help to understand how the interests of this journal s authors changed over time , particularly in the most recent time . the articles in pathology & experimental medicine ranged from more than 40% to about 20% , having been very numerous in 2013 , while progressively decreasing in 2014 and 2015 ; ; most of these papers were focused on the detection of tumor markers , but several were aimed at elucidating the mechanisms of carcinogenesis and tumor progression . there was steady interest for developmental investigations and stem cell biology with about 15% of the published articles in the last couple of years : molecular markers of cell differentiation were used to describe the process of placenta formation and organogenesis , and to phenotype stem cells . extracellular matrix and hard tissues have become increasingly important topics , demonstrating that histochemistry may be suitably applied in particular to investigate cartilage or dentin during the histogenetic process and in pathological conditions ; some papers were devoted to the temporomandibular joint in healthy and diseased subjects , while other proposed in vitro models for chondrogenesis or dentinogenesis . techniques for culture in vitro were also used for tissue engineering , using different supports for growing and differentiating various cell types : histochemistry thus proved to be an effective mean to characterize the structural and functional features of cells to be used for reconstructive medicine . the incidence of articles on neurons & muscle has progressively decreased from 2013 to present , although a series of interesting article was published especially for muscle cells . in particular , it was observed that both the ultrastructural organization and the nuclear function of myonuclei and satellite cells nuclei in the skeletal muscle from patients with myotonic dystrophy were similar to those in the muscles from sarcopenic individuals , suggesting that similar nuclear mechanisms may lead to skeletal muscle wasting . there was a steady and progressive rise in the percentage of papers presenting new methods or improved techniques : they were about 12% of the published articles in 2013 growing to more than 20% in 2014 and 2015 . actually , this is consistent with the scope of the european journal of histochemistry , which has always been an open forum for scientists to present new methods and techniques , and to discuss their experimental results , as it recently occurred for the molecules responsible for some characteristics of cell autofluorescence . in the attempt to localize specific molecules at high resolution , ultrastructural cytochemistry proved to be especially suitable , and demonstrated by the several papers published : in particular , diaminobenzidine photo - oxidation was effectively used to visualize the endocytotic pathways after staining the plasma membrane with a fluorescent dye , and to simultaneously detect immunogoldlabeled antigens at the high resolution of transmission electron microscopy . as a journal of functional cytology , the european journal of histochemistry has traditionally published many papers on cell and tissue biology in a variety of vertebrate and invertebrate species . after a relative decrease in 2013 and 2014 , in the present year more than 20% of the published papers were on these subjects , thus demonstrating that histochemical techniques are powerful tools for properly describing cell and tissue organization as well as functional microanatomy in different taxa of still poorly described organisms . in a comparative perspective , the histochemical evidence , in parallel with molecular data on protein and dna , may help to elucidate the origin and evolution of cell and tissue physiology . the overall influence of an academic journal on the scientific community is usually estimated by the impact factor , which is a measure reflecting the mean number of citations obtained by recent articles published in a given journal . this index does not take into account the subject category of the journals where the citing articles were published ; but especially for journals devoted to a variety of subjects and techniques , as the european journal of histochemistry is , it should be interesting to consider this aspect , too . the more than 200 articles published in the period 2011 - 2015 in this journal have been cited in 340 different periodicals . figure 3a shows the percentage of citations in these journal which have been grouped according to their main topic and their first subject category reported in the thomson reuters journal citation reports . as expected , the journals on histochemistry were those in which the articles published in the european journal of histochemistry were most frequently cited . in agreement with the evidence in figure 1 and 2 , a large fraction of the citations were in medical journals ( dealing with both general and experimental medicine , and with oncology ) , and in journals on cell and tissue biology or on biochemistry and molecular biology . in most of the articles published in the european journal of histochemistry , specific molecular complexes were detected , and their structural location in cells and tissues was related with function . in addition , several papers also described new methods or presented improvements of well - established techniques thus providing new opportunities for application to a wide variety of research subjects . it is therefore not surprising that citations are frequently found in journals of neurobiology , anatomy & morphology , pharmacology & toxicology , reproductive biology , veterinary sciences , physiology , endocrinology , tissue engineering & biomaterials , plant cell biology , as well as in multidisciplinary journals ; significantly , the citations in histochemical journals of the articles published in 2011 and 2012 progressively decreased from 2013 to 2015 , while increasing during the same timespan in the journal of medicine , pharmacology , physiology and tissue engineering ( figure 3b ) . in the last couple of decades , histochemistry has become a true molecular biology in situ , aimed at detecting single molecules in the very place where their functional role is exerted . the potential of histochemistry for diagnosis and prognosis has greatly increased by the use of refined techniques for investigating gene expression in situ . vital histochemistry ( chiefly , enzyme histochemistry ) provides evidence of the biochemical events driving and controlling cell physiology . the recent technological improvements in super - resolved light microscopy and in electron microscopy as well as the progress in correlative microscopy , and in imaging molecules in vivo allowed to shed light on the molecular processes responsible for cell differentiation or which account for the onset a pathological condition . it is easy to foresee that , in the future , the histochemical journals will ever more be an attended forum for basic and applied scientists in the biomedical field . but a journal of histochemistry should also be open to an audience as varied as possible within the biological area , publishing articles on organ , tissue and cell biology in very different experimental models . this open - minded attitude will keep histochemistry fresh and vital , and will also be beneficial for histochemical journals : offering hints for the applications of histochemical techniques also to non - histochemist scientists will expectedly increase the number of potential authors , thus expanding the research horizon to novel and possibly exclusive subjects . candidemia and invasive candidiasis ( c / ic ) show an increasing incidence in the nosocomial setting . the crude mortality of those infections ranges between 36%63% depending on patient population.14 comorbidities , aging and age - associated physiological changes , higher rates of oropharyngeal colonization with candida species , and concomitant drug use make elderly patients ( > 65years old ) more vulnerable to infections . for this reason fungal infections have become a major problem in older adults , since age is a well - documented predisposing factor with increased impact on mortality.57 the cutoff age for the elderly population can not be clearly defined because aging is a continuous process . it is also clear that aging is a multifactorial process influenced by both genetic and environmental parameters . many studies dealing with candida infection in the elderly have used several different arbitrary cutoff points such as over 60 , 65 , or 70 years old . however , most prospective epidemiological studies define elderly population as the age group > 65 years.59 the elderly population is large and is growing in proportion to the general hospitalized population , but available data on epidemiology , clinical impact , and outcome of nosocomial fungal infections are limited.710 the indications for antifungal therapy are the same for older as well as younger individuals , and the initial antifungal therapy should be selected on the basis of the infecting organism and the local epidemiology.9,11 fluconazole is generally effective against c / ic but its use may be limited by the increasing prevalence of candida species ( spp . ) with acquired or intrinsic resistance . echinocandins are recommended as first - line treatment for c / ic in all patients , and more specifically in hemodynamically unstable patients or in those with prior azole exposure , or for invasive infections caused by c. krusei or c. glabrata because of their activity against azole - resistant strains , while amphotericin b remains the cornerstone of antifungal treatment.1,9,1115 in this review , we aim to discuss the management and treatment options of fungal infections in the elderly population , considering additionally the specific conditions and the impact of potential comorbidities and drug interactions . invasive candidiasis ( ic ) ( also called systemic ) , is the invasion of candida spp . in a human organ ( invasion via the bloodstream is called candidemia ) . if multiple organs such as the brain , heart , kidneys , lungs , and liver are affected , the condition is called disseminated candidiasis . are ubiquitous ( more than 200 species have been described ) ; most consist partly of the human microbiological flora , although only 10% of these species are known to be responsible for infections in humans.16 indeed , at least 17 different candida spp . cause ic in humans ; c. albicans is the most common worldwide , presenting a global average of 66% of all candida spp . , with large geographical discrepancies according to the artemis disk global antifungal surveillance study.17,18 candida spp . account for 8%10% of all nosocomial infections , next to coagulase - negative staphylococci ( 31% ) , staphylococcus aureus ( 20% ) , and enterococci ( 9% ) and have become the fourth most common cause of hospital - acquired bloodstream infections ( bsis ) in the usa.19 other studies in europe and canada , with the exception of denmark , reported a lower incidence of candidemia than that reported in the usa ( 1.9% in finland , 4.9% in iceland and spain ) , reflecting probable differences in patient demographics and comorbidities , as well as in medical practices and/or diagnostic methods.6,20 in all of these studies the highest incidence occurred at the extremities of the age spectrum ( infants of less than one year old and in adults > 65 years old).6,2124 candida albicans is the most common pathogen causing ic worldwide , though a shift towards non - albicans species has been noted over the years . c. glabrata represents an important fungal pathogen , ranking second to c. albicans as a cause of bloodstream infection.6 in the usa , c. glabrata accounts for 20%24% of all candida bsis but in other geographical regions , including europe , lower rates have been reported.6 the origins of these discrepancies are unclear but previous exposure to azoles , increasing patient age , presence of underlying diseases such as malignancies , and different geographic locations or technical methodologies regarding blood cultures older patients ( > 65 years ) have increased risk of candidemia due to c. glabrata and increased risk of dying ( 29% ) as well.25 systemic broad spectrum antibiotic use , central venous catheters , long stay in the intensive care unit ( icu ) , renal insufficiency , and total parenteral nutrition were identified as the most important risk factors . it is unclear whether the higher identified rate of oropharyngeal colonization with c. glabrata in older , compared to younger , adults , is related to candidemia.26,27 blot et al have studied the outcome of critically ill patients with candidemia and found that fungemia from c. glabrata was significantly associated with older age . older age , polymicrobial bloodstream infection and acute renal failure were independent predictors of mortality.11 a significant epidemiological shift towards c. glabrata as a cause of candidemia has been reported in oncology centers , compared to individual hospitals.6,25,2830 c. paraspilosis is an exogenous pathogen , found mostly on skin rather than mucosal surfaces , and is known for its ability to form biofilm on catheters and other implanted devices . it is spread through hand contamination in hospitals and nursing homes . about 38% of c. paraspilosis bsis are acquired outside the hospital , a finding consistent with the fact that older patients often receive home health care with indwelling catheter use due to various chronic diseases.26,3133 bsis due to this species are associated with a lower mortality rate than other candida spp.2,6 c. tropicalis is an important pathogen in neutropenic patients with hematologic malignancies and mucositis . this pathogen is very common in latin america , responsible for 22% of bsi isolates but , according to the artemis disk global antifungal surveillance study , its incidence worldwide has reached 4%7% with an increasing trend.18 c. krusei is another important pathogen among patients with hematologic malignancies , and among blood marrow transplant recipients , characterized by intrinsic resistance to fluconazole.1 c. krusei accounts for 2%5% of all candida infections worldwide , having emerged in oncology patients under prophylaxis with fluconazole.28,34 interestingly , it has been reported that exposure to piperacillin - tazobactam and vancomycin leads more often to c. krusei infections than does exposure to fluconazole , because the former drugs promote skin and gastrointestinal colonization , rendering the human host more vulnerable to c. krusei bsis.35 c. guillermondii and c. rugosa are common in latin america and responsible for clusters of hospital infection , exhibiting low susceptibility to fluconazole , while they are considered as rare causes of catheter - related candidemias in other countries.3639 c. incospicua and c. norvegensis are both phenotypically similar to c. krusei , exhibiting intrinsic resistance to fluconazole , causing candidemia in human immunodeficiency virus - infected patients and in patients with hematologic malignancies . c. norvegensis has been found mostly in respiratory specimens.4042 the aging process leads to variable changes in physiological and morphological functions , rendering older patients potentially more vulnerable to infections , particularly from fungal species ( table 1).7 aging leads to hyposalivation , which in turn alters the normal microflora of the oral cavity , so that it has fewer anaerobic bacteria , such as enterococci.43 less saliva production limits peptide and protein presence in the oral cavity , and the lack of substances with broad antimicrobial activity , such as lysozyme , contributes to oral candidiasis.43 in the supragingival plaque , which is responsible for caries formation , candida species are the predominant pathogens , especially in adults > 70 years old.44 moreover , in dental prostheses various candida species can be found , with c. albicans being the most prevalent , followed by c. glabrata and c. tropicalis.44 this colonization is further influenced by ( a ) poor oral hygiene , ( b ) drugs that irritate or damage the oral mucosa , such as cytostatics , ( c ) drugs that alter the oral flora synthesis , such as antibiotics , or ( d ) by concurrent diseases , such as iron deficiency anemia . once the oral cavity is colonized , it is easier for the yeasts to reach the respiratory system , and since candida is a commensal of the gut lumen and the cutaneous surfaces , the colonization index is increasing.43,45 candida spp . are common in the urine of the elderly , especially after treatment with broad spectrum antibiotics . differentiating asymptomatic candiduria , even in high concentrations ( > 10/ml urine ) , from a true infection which triggers a systematic inflammatory response is difficult , and treatment is influenced by the biofilm formation in the urinary catheter.43 the biofilm formation is an aggregate of microorganisms where cells adhere to each other on a surface . it may become a problem in patients with indwelling catheters , such as older people in hospitals or nursing homes . candida colonization is one of the main reasons why older people are so prone to bloodstream infections , but not the only reason . older age is always accompanied by normal physiological alterations and/or various metabolic disorders or neoplastic diseases , which disrupt the mucosal and cutaneous barrier and make the organism more vulnerable to candida infection ( table 2).6 thus , selection of a suitable drug is based not only on the specific microorganism , and on the clinical condition and its severity , but also on all the underlying pathophysiological characteristics of the patient s advanced age . aging is characterized by diminished immunological response to infection , especially due to functional insufficiency of monocytes and macrophages , which leads to inadequate phagocytosis.43 other antigen presenting cells , such as dendritic cells , are lacking , and so are naive t - cells due to thymus gland involution.46 mature t - cells lose their memory capacity and exhibit poor and/or altered cytokine production.46 moreover , the number of circulating b - cells is diminished and their response to antigenic challenges through immunoglobulin production is weaker.47 animal studies on the aging liver have shown modifications of the hepatic physiology which affect drug metabolism . possible mechanisms , occurring normally with aging , involve reduction in total liver mass , hepatic blood flow , and protein synthesis . these factors compromise drug metabolism , such as hydroxylation , dealkylation , and reduction ; reactions occurring in phase i drug metabolism , performed by microsomal cytochrome p450.48,49 phase i is necessary to prepare the drug or toxin to undergo phase ii metabolism ( conjugation , acetylation , and methylation ) , altering its form and promoting its effective excretion.49 renal function is also impaired with advanced age . this is not reflected in serum creatinine because a 25% rise in serum creatinine level actually represents a substantial fall in gfr , probably as much as 50% , due to the exponential rise in creatinine level with declining renal function.50 this decline of renal function is often underestimated , since serum creatinine is dependent on muscle mass , which also attenuates with age and remains almost normal . therefore , older and very sick patients , with a normal creatinine value , have a gfr of only 30% of that of a young , healthy adult . this is associated with serious clinical problems with drugs dependent on renal excretion.50 polypharmacy in the elderly is another important issue , relevant to both adverse effects and drugs interactions . a recent dutch study demonstrated that almost 75% of the elderly population was being treated with at least 4 drugs , suggesting that elderly patients are not only prescribed a greater number of medications than younger patients , but they also receive drugs in a more inappropriate manner.5153 analgesics , including nonsteroidal anti - inflammatory drugs ( nsaids ) ( eg , acetylsalicylic acid , ibuprofen , indomethacin , naproxen ) , narcotics ( eg , hydrocodone ) , non - narcotic pain medications ( eg , acetaminophen ) , or drugs with other mechanisms of action that act synergistically on pain relief ( benzodiazepines , tricyclic antidepressants ) , comprise the most popular drug categories.53 furthermore , polypharmacy involves , apart from pain relief , treatment of other diseases , such as hypertension , diabetes , chronic obstructive pulmonary disease , heart failure , or cancer . moreover , alterations in body composition such as decrease in total body water or increase in body fat may result in unexpected toxic effect or duration of action of various drugs . the prevalence of the effect of drug drug interactions on the liver is > 74% in older women of which 63% involve nsaid use.54 apart from multiple drug use , the mechanisms of drug - induced liver injury in the elderly include gender , dosage and treatment duration , drug formulations , nutritional status , genetic susceptibility , environmental factors ( eg , alcohol abuse ) , and underlying comorbidities.53 invasive candida infections include clinical syndromes of different severity where the diagnosis is a challenge , especially in critically ill , immunocompromised , or elderly patients . the signs and symptoms vary from silent or atypical , to that of a bacterial infection . newer culture methods have raised the sensitivity of candida detection to almost 70% , but it takes a minimum of 24 to 48 hours to become positive and this may come late in the course of the infection.55 moreover , patients are often under fluconazole prophylaxis which may render the cultures negative at time of testing.56 two antigen based tests are currently available for the early diagnosis of candidemia , relying on detecting components of the fungal cell wall . the first method detects mannan levels , which is a major component of the candida cell wall . in high risk patients , it is recommended to be performed two to three times per week , since its circulation in the bloodstream is intermittent . sensitivity and specificity of this test , when combined with anti - mannan antibodies detection in critically ill but not immunocompromised patients , are 83% and 86% respectively.5659 the second diagnostic tool is based on the detection of 1,3--d - glucan . this test has been evaluated mostly in critically ill patients and has demonstrated an overall sensitivity of 77% and specificity of 85% for subjects with proven or probable ic.5860 one single positive test is indicative of the infection , but it must be interpreted with caution due to false positive results.55 a negative 1,3--d - glucan test is associated with high negative predictive value ( > 90% ) and can be used to rule out ic , especially in patients with neutropenia.6062 finally , nucleic acid - based detection methods ( real - time polymerase chain reaction ) have been developed for five different candida spp . although these techniques have shown significant advances in the early and specific diagnosis of ic , further evaluation must be conducted in specific populations such as the elderly , considering the relatively weak immune systems and variable immunological responses among this age group , which render the diagnostic accuracy of the above methods less precise.55 the management of candida infection includes prophylactic , preemptive , empiric , and targeted treatment ( figure 1 ) . preemptive therapy is justified in the presence of positive inflammatory markers biomarkers in conjunction with certain predisposing risk factors . empiric therapy is warranted in patients with a currently unknown infection , for whom treatment is justified based on clinical judgment , while targeted treatment is administered when the diagnosis of a certain pathogen is documented . currently available drugs against ic include amphotericin b ( amb ) and its derived lipid formulations ( lfamb ) , azoles ( triazoles ) including fluconazole ( flu ) , voriconazole ( vor ) , posaconazole ( pos ) , and itraconazole , and echinocandins including . are susceptible to these agents , except for those with intrinsic or acquired resistance after exposure to other drugs . amb is a polyene macrolide antifungal regimen with fungicidal action , which was considered in the past to be the gold standard for the treatment of invasive fungal infections.62 derived from streptomyces spp . , it has a high affinity for the sterols of fungal and bacterial membranes , forming small transmembrane channels which lead to monovalent ion leakage and cause fungal cell death . derivatives of amb were developed in order to limit toxicity , especially renal failure , which has been rated between 49%65%.50,6365 three lipid formulations of amb ( lfamb ) are commercially available , all with a good fungicidal activity and no differences in efficacy . the formulations include a true liposome structure lfamb ( ambisome , gilead , cambridge , uk ) ; a ribbon - like structure amb lipid complex ( abelcet , sigma - tau pharmaceuticals , inc , gaithersburg , md , usa ) ; and a colloid dispersion , amphotericin b colloidal dispersion ( abcd ) ( amphocil / amphotec , evaluatepharma , london , uk ) with a disc - like structure . all lipid formulations have less nephrotoxicity than conventional amb - deoxycholate.6466 among these , the liposome structure of amb seems to have lower nephrotoxicity rates ( 15% ) than the ribbon - like structure ( > 40%).67 lfamb is used in a dose of 3 to 5 mg / kg daily in life - threatening mycoses , as well as for empirical treatment of suspected ic , or in situations where an antifungal agent with rapid time - kill rate and high post - antifungal effect is needed.11,68 however , in older patients it should be used with caution , especially if parameters such as dehydration , large cumulative dosage , abnormal baseline renal function , and concomitant nephrotoxic drug use exist . the azoles flu , itraconazole , vor , and pos inhibit the fungal cytochrome p450 enzyme 14-demethylase and prevent the conversion of lanosterol to ergosterol , which is essential for the fungal cell membrane integrity . all the above azoles demonstrate activity against candida spp . but reduce activity against c. glabrata and c. krusei . flu , with primarily fungistatic effect ( 800 mg or 12 mg / kg loading dose , followed by 400 mg or 6 mg / kg daily ) , has comparable efficacy to amb for the treatment of candidemia , while it is indicated as empirical and curative treatment in non - neutropenic patients.6970 flu is recommended for the treatment of c. parapsilosis bsi , while among all the triazoles , it possesses the greatest penetration to the cerebrospinal fluid and it is therefore indicated for the treatment of central nervous system and intraocular infections . prophylactic use of flu led to a shift to resistant candida species , and its use alters ( and may even increase ) the frequency of infection due to molds . prophylaxis ( 6 mg / kg daily ) is recommended in solid organ transplant recipients ( liver , pancreas , and small bowel ) , during induction chemotherapy and in stem cell transplant recipients during the period of neutropenia.11 flu is an inhibitor of the human cytochrome p450 system and therefore it decreases the metabolism or increases the concentration of any drug metabolized by these enzymes . this should be kept in mind , considering the high number of concomitant drugs that older people take , which also undergo hepatic metabolism , in order to avoid serious and life - threatening drug drug interactions . serum levels of warfarin , phenyntoin , or oral hypoglycemic agents are increased by the azoles , whereas serum digoxin levels may increase . another rare adverse event is the potential effect on electrocardiographic qt interval , whose elongation increases the risk of ventricular arrhythmias , especially if drugs are used concurrently , which also prolong its duration ( eg , macrolides , fluoroquinolones , anticholinergic , antihistamines , diuretics , and the gastroprokinetic agent cisapride ) . itraconazole has a broader spectrum of activity than flu ( in vitro activity against candida spp . , aspergillus spp . , and dimorphic fungi , but not as broad as vor or pos ) , but is not able to penetrate cerebrospinal fluid . , while it is active against c. krusei , c. guillermondii , and c. lusitaniae.71 intravenous vor is complexed to a cyclodextrin molecule and after two loading doses of 6 mg / kg every 12 hours , a lower maintenance dosage of 34 mg / kg twice daily is recommended . due to cyclodextrin accumulation , vor is not indicated in patients with renal dysfunction and creatinine clearance of < 50 ml / min.72 vor is effective in candidemia , but it offers little advantage over flu and is therefore recommended as a step - down oral therapy for c. krusei infection and for flu - resistant , vor - sensitive c. glabrata infection . oral vor does not require dosage adjustment in renal insufficiency , but is the only triazole that requires dosage reduction in patients with mild - to - moderate hepatic insufficiency . in a randomized , international , multicenter trial comparing vor with lfamb as empirical antifungal treatment , the authors suggested that vor could be a suitable alternative to lfamb in patients with neutropenia and persistent fever.73 pos is available only as an oral suspension with high oral availability and seems to be more active than the other triazoles . pos exhibits a broad spectrum activity against yeasts , molds , or rare fungal strains . echinocandins ( ecs ) are a new class of antifungal agents that target the fungal cell wall by inhibiting 1,3--d - glucan synthetase , leading to osmotic instability and cell death . the three members of the group , cfg ( loading dose of 70 mg , then 50 mg daily ) , mic ( 100 mg daily ) , and anidulafungin ( loading dose of 200 mg , then 100 mg daily ) , are all available only for parenteral use . each of these agents has been studied for the treatment of ic in comparative and noncomparative clinical trials.7477 the mics of the echinocandins are low for a broad spectrum of candida spp . , including c. krusei and c. glabrata . c. parapsilosis demonstrates less in vitro susceptibility ( higher mics ) than most other candida spp . , and that has raised the concern of its being less responsive.71,78,79 similarly , there have been reports of increased clinical failure and persistence of infection with this species , claiming that c. parapsilosis infection may indeed require higher echinocandin dosage.1,80 therefore , in the recent clinical practice guidelines for the management of candidiasis , flu is the treatment recommendation for c. parapsilosis infection , unless the patient has already received an echinocandin , is clinically improved , and has negative follow - up cultures.11 until now , this matter was still under debate , since a recent meta - analysis showed that ecs are effective for the treatment of candidemia or invasive candidiasis due to c. parapsilosis.81 another important issue , unique for the ecs , is the eagle effect , a term used to describe the paradoxical in vitro and in vivo growth of candida and aspergillus isolates when the dose of the drug gets over the mic level.82 this phenomenon has similarities to the eagle effect observed in other cell wall active antimicrobial agents , such as penicillins . although the clinical impact of this phenomenon has not been elucidated , it might be of some importance in biofilm tretment.62 none of the ecs require dosage adjustment for renal insufficiency or dialysis . both cfg and mic undergo minimal hepatic metabolism , but neither is a major substrate for cytochrome p450 and therefore they have minimal drug - drug interactions . anidulafungin has not hepatic metabolism ; it undergoes slow chemical degradation to a ring opened peptide with no antifungal activity . though concerns have been raised about the potential hepatotoxicity of mic due to tumor formation in rodents , cfg is the only ec for which dosage adjustment is recommended for patients with moderate to severe hepatic dysfunction . the clinical practice guidelines favor the use of an ec as initial therapy for candidemia in non - neutropenic as well as neutropenic adult patients , with moderate to severe illness.11 alternatively , flu and lfamb may be used , but for infection due to c. glabrata an ec is preferred , since the triazoles have diminished activity against this species . moreover , very little is known about the pharmacological / pharmacokinetic properties of antifungal drugs in the elderly . the diminished drug clearance that occurs naturally with aging , along with the presence of other comorbidities and drug use , make the pharmacodynamic and pharmacokinetic issues very intriguing . drug interactions and comorbidities are the main reasons why we would not recommend azoles or amphotericin b as our first therapeutic choice in this specific population . dose modification is not warranted unless indicated for other reasons ( eg , hepatic or renal dysfunction).83 monitoring of plasma levels could be an option , but it is time and resource consuming , even in health care facilities where the method is available . monitoring drug levels in plasma can not be indicated for routine use unless future studies provide us with more data . flucytocine ( or 5-fluorocytosine , 5fc ) is an antimetabolite that acts as an antifungal against candida spp . 5fc enters the fungal cell via cytosine permease , and is metabolized to 5-fluorouracil , which is incorporated extremely closely into the fungal rna , inhibiting both dna and rna synthesis . most of the drug is excreted unchanged in the urine , so that dose adjustment is necessary for patients with renal dysfunction . considering the fact that 5fc is rarely administered as a single agent but in combination with other antifungal drugs ( mainly lfamb ) for patients with ic , it is not suggested as a combination in elderly patients due to the accumulative nephrotoxicity risk . nosocomial candidemia is associated with increased mortality and this seems to be further aggravated in case of delay in antifungal drug initiation.8486 morrell et al have shown that initiating empiric antifungal treatment more than 12 hours after the first blood culture sample is associated with a greater risk of hospital mortality than when patients are started on antifungal therapy within the first 12 hours.84 in a 5-year study , parkins et al studied 207 patients with ic ; 64 patients ( 32% ) received empirical therapy , in 51 ( 26% ) of which was deemed appropriate.86 similarly , kumar et al demonstrated a 12% decreased survival probability for every hour s delay in patients with fungal septic shock.87 therefore , prompt initiation of early empiric therapy is warranted in high risk patients . knowledge of risk factors for ic may help to identify those patients who could benefit from early antifungal therapy ( table 3 ) . the candida score was first introduced by len et al and the epcan study group , from data available from the surveillance study of fungal infection and colonization in critically ill patients.88 clinical sepsis ( 2 points ) , multifocal colonization ( 1 point ) , surgery ( 1 point ) , and total parenteral nutrition ( 1 point ) are the risk factors that must be evaluated by the physician in order to identify patients who are candidates for empirical treatment . a score of > 2.5 showed 81% sensitivity and 74% specificity for the early administration of empirical treatment in icu patients.88,89 in summary , the increase in invasive candidiasis in older adults has become an important clinical problem , since the older population is growing and is nowadays more likely to take aggressive chemotherapeutic regimens for cancer , or immunosuppressive drugs for nonmalignant diseases . moreover , aging leads to variable physiological changes , rendering older patients potentially more vulnerable to fungal infections . elderly patients are more easily colonized by pathogenic fungi and have an increased incidence of c. glabrata fungemia , which has higher mortality rates as well as higher rates of resistance to fluconazole , especially after exposure to the drug . therefore , although clinical manifestations in older and younger adults may be similar , for the treatment of the former the use of an echinocandin is safer , since treatment with amphotericin b is associated with increased nephrotoxicity risk . azoles are less toxic but they must be used with caution , since older adults are usually under a number of medications and the risk of serious drug

especially in recent years , biomedical research has taken advantage of the progress in several disciplines , among which microscopy and histochemistry . to assess the influence of histochemistry in the biomedical field , the articles published during the period 2011 - 2015 have been selected from different databases and grouped by subject categories . as expected , biological and biomedical studies where histochemistry has been used as a major experimental approach include a wide range of basic and applied researches on both humans and other animal or plant organisms . to better understand the impact of histochemical publications onto the different biological and medical disciplines , it was useful to look at the journals where the articles published in a multidisciplinary journal of histochemistry have been cited : it was observed that , in the five - years period considered , 20% only of the citations were in histochemical periodicals , the remaining ones being in journals of cell & tissue biology , general and experimental medicine , oncology , biochemistry & molecular biology , neurobiology , anatomy & morphology , pharmacology & toxicology , reproductive biology , veterinary sciences , physiology , endocrinology , tissue engineering & biomaterials , as well as in multidisciplinary journals . it is easy to foresee that also in the future the histochemical journals will be an attended forum for basic and applied scientists in the biomedical field . it will be crucial that these journals be open to an audience as varied as possible , publishing articles on the application of refined techniques to very different experimental models : this will stimulate non - histochemist scientists to approach histochemistry whose application horizon could expand to novel and possibly exclusive subjects . fungi are major causes of infections among immunocompromised or hospitalized patients with serious underlying diseases and comorbidities . candida species remain the most important cause of opportunistic infections worldwide , affecting predominantly patients over 65 years old , while they are considered to be the fourth most common cause of nosocomial bloodstream infections . the rapidly growing elderly population has specific physiological characteristics , which makes it susceptible to colonization and subsequent infection due to candida species . comorbidities and multidrug use should be taken into account any time the therapeutic regimen is under consideration . different classes of antifungal drugs are available for the treatment of invasive fungal infections but echinocandins , apart from their activity against resistant strains ( candida glabrata and candida krusei ) , seem to be safe , with limited adverse events and minimal drug drug interactions in comparison to the other regimens . therefore , these agents are strongly recommended when dealing with elderly patients suffering from an invasive form of candida infection .

percutaneous transluminal coronary angioplasty was introduced by gruntzig in the late 1970s as an alternative to coronary artery bypass graft surgery for coronary revascularization.1 since then , percutaneous coronary intervention ( pci ) has been accepted as a safe , reliable , and effective treatment for coronary artery disease , and its use has spread worldwide . nevertheless , in - stent restenosis ( isr ) , a complex phenomenon resulting in renewed symptoms , need for re - intervention , and poor patient outcome remained for many years the achilles heel of pci.2 the introduction , a decade ago , of first - generation drug - eluting stents ( dess ) transformed the practice of pci by drastically reducing the rate of this complication.3 the efficacy of dess has largely been demonstrated in large randomized trials , leading to their current widespread use in clinical practice . even in high - risk populations , isr incidence does not currently go above 5%10%.46 however , major concerns regarding the long - term safety of these first - generation dess have progressively arisen , especially the increased risk of late / very late stent thrombosis ( st)714 and the need for prolonged dual antiplatelet therapy ( dapt ) with an inherent increase of bleeding complications . aside from patient- and lesion - related factors , delayed re - endothelialization and recovery of endothelial function after stenting as well as inhibition of vascular repair after des implantation , all of which promote inflammation and thrombotic pathways , have been implicated in the pathophysiology of late / very late st . of note , stent polymer has also been cited as one of the main causes of these late events . in fact , the persistence of a durable polymer after complete release of the anti - proliferative drug has been shown to be responsible for local hypersensitivity and inflammatory reactions.15 these safety concerns prompted additional research , new trial design , and development of new - generation dess to reduce the rate of this rare but critical event . apart from the progress in stent platforms ( thinner struts and stent designs ) , recent research in this field has subsequently been focused on the development of new more biocompatible durable polymers or completely biodegradable polymers . third - generation dess using biodegradable polymers , like the biolimus - eluting stent ( bes ) ( nobori ; terumo corporation , tokyo , japan ) , have been developed to overcome the long - term adverse vascular reactions related to the durable polymer . in this article we then discuss the results of recent publications investigating the safety and effectiveness of the use of the third - generation bes ( nobori ) for the treatment of coronary artery lesions . the two major complications of pci , isr and st , have always been the trigger for new stent development.16 indeed , despite the fact that these events are multifactorial , stent structure has been suggested to be one of the leading causes of isr and/or st.1721 isr physiopathology has not yet been fully understood . barotrauma induced by pci is responsible for endothelial denudation and sub - intimal hemorrhages , leading to a local inflammatory response . this inflammatory process induced by vascular damage is thought to be one of the main contributors to the development of restenosis , by promoting vascular smooth muscular cell proliferation and extracellular matrix formation , resulting in neointimal hyperplasia . beside these mechanical factors , other factors have been identified as predictors for isr , including patient - related ( eg , diabetes mellitus , smoking , and renal failure ) and lesion - related ( eg , minimal lumen diameter after pci , severe calcifications , chronic total occlusions , tortuous vessel , and long lesion length ) factors.22 as mentioned above , isr remained the achilles heel of pci until the large use of dess , which were specifically developed to overcome this complication . before the introduction of bare - metal stents ( bmss ) , up to 50% of the patients treated by pci experienced restenosis . even in the bms era , isr remained one of the major limitations of this technique , with an average incidence of 20% , but that could increase up to 35% in complex lesions and diabetic patients.4,5 the introduction , 10 years ago , of first- and then second - generation dess transformed the practice of pci by drastically reducing the incidence of this complication to less than 10%.3 dess prevent restenosis by inhibiting vascular smooth muscle proliferation.2331 unfortunately , they also delay re - endothelialization of stent struts , leading to the potential risk of late / very late st and thereby the need for prolonged dapt . since the appearance of dess , st has become the major safety concern in contemporary pci practice . st is a rare adverse event ( 1% at 1 year and then 0.5% per year ) but remains associated with high morbidity and mortality rates.32 the overall prognosis is poor : most patients in whom st occurs present with stemi ( st - segment elevation myocardial infarction ) or out - of - hospital death , and up to 30% of those who arrive alive at hospital die within the first month . numerous factors have been implicated in st physiopathology , but studies have also shown that these predictors vary over time . these data highlight the complex physiopathology of st , depending on the timing of event occurrence . acute ( within 24 hours ) and early st ( within 30 days ) are likely related to mechanical issues concerning the stent ( eg , minimum stent area and suboptimal stent expansion ) , inadequate platelet inhibition , or patient prothrombotic factors.33 late ( up to 1 year ) and very late st ( after 1 year ) have been attributed to incomplete vascular healing and/or inadequate neointimal coverage , which in turn , promote inflammation and activation of thrombotic pathways15 and late or acquired stent malapposition . dapt associating aspirin with an oral p2y12 inhibitor has been shown to be the standard therapy following coronary stenting in order to significantly reduce cardiac events , especially st after pci.34 current guidelines support the use of dapt for 612 months after des implantation.3539 in 2006 , the potential risk of late / very late st after des implantation raised the question of prolonging dapt even beyond the first year.4041 however , prolonged dapt has also clearly been associated with an increased risk of bleeding.4247 availability of new biodegradable polymers and/or stents may shorten the duration of necessary dapt and therefore minimize the risk of major bleeding to which it is associated . first- and second - generation dess have three major components : the stent platform , the antiproliferative drug , and the polymer . all of these factors have been subject to modification and have become a target for research and development . it provides the radial force to prevent vessel occlusion provoked by vessel injury following pci . cobalt - chromium and later platinum - chromium platforms used in second - generation dess permitted similar stents radial strength all the while enabling a thinner strut design and subsequently significantly improved deliverability and a reduced rate of isr.4849 unfortunately , the presence of a permanent scaffold in the vessel constitutes a stimulus for platelet aggregation and may lead to st in patients with nonoptimal antiplatelet therapy and/or incomplete stent endothelialization . recently , bioabsorbable platforms that biodegrade over a period of months have been developed , with the purpose of allowing the restoration of a normal vascular physiology and function over time . ultimately , no foreign material is left exposed in the bloodstream . these stents may also potentially preserve reactive vasomotion and permit expansive remodeling.50 there are several antiproliferative drugs with different modes of action . the goal of these drugs is to inhibit vascular smooth cell proliferation and migration , without affecting endothelial regeneration , and have anti - inflammatory / anti - thrombotic properties . inhibitors of the mammalian target of rapamycin ( mtor ) are the dominant class of anti - proliferative drugs used for dess . the mtor inhibitors are cytostatic drugs resulting in arrest of the cell cycle at the g1 phase . aside from mtor inhibitors , tacrolimus , which acts as a calcineurin inhibitor , has also been used in dess and is a cytostatic agent with both antiproliferative and anti - inflammatory activities . finally , paclitaxel is a taxan drug , which acts as a cytotoxic drug through the stabilization of microtubules . however , all these antiproliferative agents have shown detrimental local effects on the vascular wall and on endothelial function recovery after stenting.11 new drugs ( biolimus , novolimus , and myolimus ) have been developed and have shown promising results.5152 compared with other mtor inhibitors , biolimus shows better lipophilicity . agents other than drugs are under investigation to limit restenosis , such as antibody - coated stents ( cd34 antibody - coated stents ) and nucleotide- or peptide - coated stents . the ultimate goal remains the inhibition of maladaptive neointimal proliferation , all the while promoting vascular healing . once drug elution has been completed , most polymers exert limited functions and act as a potential trigger for local inflammation and hypersensitivity and subsequently late / very late st . they promote an inflammatory response and eosinophilic infiltration in the arterial wall , causing hypersensitivity reactions and endothelial dysfunction responsible for delayed healing and lead to st.11,12,22,53 the biocompatibility , composition , formulation , degradation delay of the polymer , pharmacokinetics of the antiproliferative agent released by the polymer , and the management of variation in polymer degradation delay have become new difficult challenges for the development of stent polymers . an optimal polymer should mimic the endothelial lining in order to prevent late thrombotic complications , thus improving stent safety . given the issue of polymer - induced inflammation and thrombosis , more biocompatible durable polymers have been developed . second - generation dess are composed of these polymers , thereby improving arterial healing and potentially reducing the rate of late ischemic events . the limited function of stent polymers once the drug is eluted has also fuelled research in biodegradable polymers . third - generation dess use polylactic acids ( poly - l - lactic acid and poly d , l - lactide - co - glycolide ) as bioabsorbable polymers . the bes with biodegradable polymer ( nobori ) is one of these third - generation dess . finally , another field of research is the development of stents that elute antiproliferative drugs without the need for polymers . these polymer - free stents could prevent the potential adverse physical effects of the polymer , leading to sustained intima inhibition , improved healing , and a lessened activation of the inflammatory / thrombotic pathways . safety , efficacy , and deliverability are the main sought - out properties for the development of new dess . an optimal combination of these components , which are in part interdependent , is necessary for enhancing stent performance . the bes stent with biodegradable polymer ( nobori ) is one of the third - generation dess ( figure 1 ) . the platform is composed of stainless steel , and the strut thickness is 112 m . it is coated with a polylactic acid polymer on its abluminal surface , which is metabolized within 69 months to lactic acid , water , and carbon dioxide through interaction with the krebs cycle . the stent elutes an antiproliferative drug , biolimus ( 15.6 g / mm ) , for up to 30 days . the coating design of the stent combined with the lipophilicity of the drug is thought to optimize local drug distribution and to reduce its release into the general circulation . at the end , the nobori stent will leave only a bms in place . the nobori stent has already been compared with first- and second - generation dess with promising results . the nobori core trial54 reported late - loss with the nobori stent at 9 months , similar to that found using the sirolimus - eluting stent ( ses , cypher ; cordis corporation , bridgewater , nj , usa ) : 0.10 and 0.12 mm , respectively ( p=0.66 ) . however , the use of the nobori stent results in better endothelial recovery , with normal coronary vasodilatation in the adjacent stent segments after implantation , contrasting with the paradoxical vasoconstriction seen with first - generation dess.55,56 the nobori i trial showed non - inferiority to and subsequent superiority of the nobori stent over the paclitaxel - eluting stent ( taxus , boston scientific , maple grove , mn , usa ) : late - loss 0.11 versus 0.32 mm , p<0.001 . moreover , this trial demonstrated a lower rate of st with the nobori stent after a 9-month follow - up.57 three recent large randomized trials have compared safety and efficacy of this third - generation des with that of first- and second - generation dess with durable polymer ( table 1).58 the sort out v trial59 enrolled 2,468 patients who underwent pci in denmark and randomized them 1:1 using the nobori and cypher stents . this trial was a multicenter , prospective , non - inferiority trial ( non - inferiority margin chosen at 2% ) comparing the bes using biodegradable polymer with the ses using permanent polymer . a total of 1,229 patients were assigned to the bes group ( 1,532 lesions ) and 1,239 to the ses group ( 1,555 lesions ) . the primary endpoint was a composite of safety ( cardiac death , myocardial infarction [ mi ] , and st ) and efficacy ( target lesion revascularization [ tlr ] ) within 9 months of stent implantation ( intention - to - treat analysis ) . there were 75% male , and only 15% of the patients had a history of diabetes . altogether , 17% had undergone a previous coronary intervention . the main target vessel location was the left anterior descending ( lad ) artery ( 40.8% ) . procedure characteristics were similar between the two groups , except for maximum stent pressure , which was significantly higher in the ses group ( 18 versus 16 atm , p<0.0001 ) . composite primary endpoint occurred in 4.1% of cases in the bes group and 3.1% of cases in the ses group ( one - sided pnon - inferiority=0.06 , and p=0.22 for superiority ) . cardiac death and tlr were not significantly different between the bes and ses groups ( 0.7% versus 1% , p=0.38 ; 3.3% versus 2.1% , p=0.07 ) . of note , st occurred more frequently in the bes group ( especially within the first month ) , with no clear explanation , and the rate of st was particularly low in the ses group : 0.7% versus 0.2% , p=0.03 . the authors concluded that the bes did not show non - inferiority when compared with the first - generation ses at 9 months . the compare ii trial60 was a multicenter , open - label , randomized , controlled , non - inferiority trial ( non - inferiority margin chosen at 4% ) and aimed to compare the safety and efficacy of the bes using biodegradable polymer with a second - generation everolimus - eluting stent ( ees ) using durable biocompatible polymer ( xience v [ abbott vascular , santa clara , ca , usa ] or prime [ boston scientific corporation , natick , ma , usa ] ) . a total of 1,795 patients ( 2,638 lesions ) received a bes , and 912 patients ( 1,387 lesions ) an ees . the primary endpoint was a composite of safety ( cardiac death and non - fatal mi ) and efficacy ( tlr ) at 12 months . there were 74% male , 21.7% diabetics , and 20% had a previous history of mi . procedure characteristics were not significantly different between the groups but the rate of non - allocated stent implantation was higher in the bes group . primary endpoint occurred in 5.2% of cases in the bes group and 4.8% in the ees group ( one - sided pnon - inferiority<0.0001 , and p=0.69 for superiority ) . there was no difference in the rates of cardiac death ( 0.8% in each group , p=0.97 ) and mi ( 2.8% in the bes group versus 2.5% in the ees group ; p=0.63 ) . the rate of definite st ( 0.7% versus 0.4% , p=0.38 ) and definite / probable st ( 0.8% versus 1% , p=0.58 ) were very low and comparable between the two groups . target vessel revascularization did not differ between the bes group ( 2.9% ) and the ees group ( 2.6% , p=0.69 ) . the authors concluded that the bes was shown to be non - inferior to the ees in terms of safety and efficacy at 1-year follow - up after pci . the 5-year follow - up of the study should answer the question of long - term safety and efficacy of the bes compared with second - generation dess . the next trial61 was a prospective , multicenter , randomized , open - label , non - inferiority trial ( non - inferiority margin chosen at 3.4% ) comparing the bes ( nobori ) with the ees ( xience v or prime ) in terms of tlr at 1 year . may to october , 2011 , in 98 japanese centers , 3,235 patients were randomized 1:1 without any exclusion criteria to undergo pci with either the bes or the ees . altogether , 30% of included patients were over 75 years old , and 46% of the population in each group were diabetic . efficacy endpoint was any tlr at 1 year , whereas the primary safety endpoint was a composite of death and mi . primary efficacy endpoint occurred in 4.2% in both the bes group and the ees group ( one - sided pnon - inferiority<0.0001 , and p=0.93 for superiority ) . the rate of any mi and stroke was similar between the two groups ( 3.3% versus 3.1 [ p=0.77 ] and 1.4% versus 1.5% [ p=0.89 ] ) . no difference was observed in terms of mortality , 2.6% and 2.5% in the bes and ees groups , respectively , p=0.9 . with regard to the rate of st , the incidence was extremely low , and there was no difference between the bes group and the ees group ( 0.25% and 0.06% , p=0.18 ) . the angiographic sub - study shows that the difference in in - segment late loss between the two groups was 0.03 mm , demonstrating non - inferiority of the bes , with a margin of 0.195 mm . of note , the rate of stent fracture was significantly higher in the bes group ( 3.1% versus 0% ; p=0.004 ) . the authors concluded in the non - inferiority of the bes over the ees in the setting of stable coronary artery disease . clinical outcomes were excellent , with a low rate of tlr and extremely low rate of st in each group . third - generation dess using biodegradable polymer such as the nobori stent have been developed to overcome long - term adverse effects observed with first - generation dess related to the use of durable polymers , and to thereby shorten the duration of dapt and the ensuing risk of hemorrhagic complications . biodegradable bess should nonetheless possess similar efficacy to actual dess in preventing isr ( lower rate of tlr as compared with bmss ) . first studies evaluating bes efficacy and safety have shown promising results , with significantly lower in - stent late loss than with paclitaxel - eluting stents , and similar in - stent late loss as sess.54,57 however , the recent compare - ii , sort out v , and next trials , all three of which were sufficiently powered to compare clinical outcomes , showed contrasting results . to date , only the compare - ii and the next trials have evaluated a new third - generation des versus a second - generation des ( xience v or prime ) . the compare - ii trial demonstrated non - inferiority of the bes relative to second - generation eess , but procedural failure was significantly higher in the bes group.60 by contrast , non - inferiority of the bes relative to the first - generation ses was not shown in the sort out v trial due to a higher risk of early st without a clear explanation for this phenomenon.59 in fact , the rate of st was particularly low in the ses group in this study . the next trial is the largest trial , to date , evaluating the efficacy and safety of the bes as compared with the ees.61 in this trial , clinical and angiographic outcomes of the bes group were non - inferior to the ees group , and the device implantation success rate was comparable in both groups . of note , a higher rate of stent fracture was seen in the bes group in the angiographic sub - study of the next trial , but its imputability in very late st or tlr is uncertain and needs further exploration . of major importance , all these trials have enrolled low risk patients , and notably a low incidence of diabetics , with relatively simple angiographic lesions ( eg , large vessels , low rate of bifurcations , and lesion length < 20 mm ) . these facts are critical regarding the interpretation of the results and the low rate of adverse events observed in these three trials : the primary efficacy endpoint was lower than 5% in all studies , and any conclusion must be interpreted with caution , especially in non - inferiority trials . in addition , a safety comparison of modern dess is challenging . indeed , st , especially very late st , is a rare complication , and although 12-month results of standard trials usually enable safety or efficacy to be assessed , longer follow - up ( 5 years or more ) and large trials are mandatory in the present case in regard of late - event exploration . the rate of st in the present studies is particularity low ( around 0.5% at 1 year ) , and these trials are not powered enough to evaluate such a low frequency event . finally , if present , the expected benefit of third - generation dess like the nobori stent over older dess would logically appear only after 1 year . to date , no very late st with the nobori stent has been reported in the literature , with the limitation that long - term follow - up is currently only available for very few patients . the leaders trial was the first randomized study to evaluate bess against durable polymer first - generation dess . this trial included higher risk patients , and the rate of events was on average twice as high as compared with the compare ii , sort out v , and next trials . the leaders study compared another third - generation bes ( biomatrix flex , biosensors , newport beach , ca , usa ) with sess . the only difference between the biomatrix flex and the nobori stent is the presence of an ultra - thin non - degradable parylene coating between the stent and the polymer on the nobori stent to assure polymer attachment to the stent struts , which do not exist on the biomatrix flex. the final 5-year report has just been published62 and demonstrates that the bes is non - inferior to the ses with regard to the primary endpoint ( cardiovascular death , mi , and clinically driven target vessel revascularization ) ( 22.3% in the bes group versus 26.1% in the ses group ; p=0.071 ) . in addition , even though no significant difference was observed between the bes and the ses in total definite st at 5 years , the bes was associated with a significantly lower rate of very late st and secondary composite endpoint ( all cause death , any mi , any revascularization ) ( 0.6% in the bes group versus 2.2% in the ses group for very late st [ p=0.003 ] , and 35.1% versus 40.4% for secondary composite endpoint [ p=0.02]).62 a recently pooled analysis based on patient individual data from the isar - test 3 , the isar - test 4 , and the leaders trial63 has demonstrated that biodegradable polymer dess ( including : biodegradable polymer bes [ biomatrix flex ] , n=857 ; and biodegradable polymer ses , n=1,501 ) improve safety and efficacy over first - generation sess during long - term follow - up . the three trials separately showed no differences between bess and dess in the past , highlighting the lack of statistical power of these studies ( taken alone ) to detect relevant differences in very low frequency events such as st . in this meta - analysis , the benefit of bess was seen in the rate of st ( 0.2% versus 1.3% , p=0.004 ) , mi ( 1.5% versus 3% , p=0.03 ) , and cardiac death ( 3.9% versus 4.9% , p=0.05 ) . of note , bess also showed a significant reduction in tlr in this meta - analysis ( 12% versus 13.7% , p=0.029 ) . these trials were performed with sirolimus first - generation dess as control , and results can not be extended to other second - generation dess . further studies using second - generation dess as control are required to make any conclusions . interestingly , such a meta - analysis of trials that have evaluated the nobori stent may carry interesting conclusions in the next future . bmss and dess have changed the landscape of current pci by significantly reducing the rate of isr . however , first - generation dess have been associated with a higher rate of late or very late st . the physiopathology of this rare but serious event is multifactorial , but inflammation and delayed arterial healing , of which the durable polymer may be a putative mechanism , has an important role . the concept of biodegradable polymer and totally bioresorbable scaffolds looks , in consequence , very interesting . long - term follow - up is mandatory to see if the implantation of these third - generation dess will improve clinical outcomes with a lower rate of st and mi . further studies and results of their long - term follow - up would definitely shed light on patient outcomes with the use of such devices . the modern concept of decompression for traumatic brain injury ( tbi ) was introduced by harvey cushing before world war i . the monro - kellie hypothesis dictates that the amount of space within the skull is constant ; therefore , when the pressure is raised death occurs by herniation when the capacity for adjustment by fluid shifts from the cerebrospinal fluid and vascular compartments are already maximized . increasing the skull size by removing bone and opening the dura delays or prevents these limits from being reached . however , in the 90 years since cushing made these observations , medical , radiographic , and surgical advances in the management of tbi have obviated the need for an aggressive surgical approach in all but a minority of cases . in spite of the ability to control intracranial pressure ( icp ) elevation in most cases with removal of mass lesions , osmotic diuretics , ventricular drainage , sedative / hypnotic agents , and prevention of hypercapnea , occasional cases occur in which icp elevation accelerates in spite of maximal conservative medical therapy , and then so - called heroic measures are employed . these are considered at the ' option ' level in the american association of neurological surgeons criteria for management of severe brain injury because no large randomized trial has proven their efficacy . in the pediatric population , ruf and colleagues as well as taylor and associates have addressed the issue of using decompressive craniectomy as a more formal part of a head injury protocol before going to other ' option ' therapies once icp elevation is affirmed . bifrontal decompressive craniectomy is an aggressive approach described by kjellberg and prieto before the era of modern neuroimaging with computed tomography . this approach is particularly useful in the pediatric population , in which diffuse injury without mass lesions and with icp elevation is relatively common . hemicraniectomy represents a large cranial and dural decompression , often associated with removal of mass lesions such as subdural hematoma or traumatic intracerebral hematoma . bitemporal decompressions are unilateral or bilateral bony decompressions designed to take the pressure off the temporal lobes to prevent uncal herniation , and have been used in other cranial conditions such as pseudotumor cerebri . gower and coworkers reported 40% mortality and 50% favorable outcome in a study of subtemporal decompression for icp control in 10 patients with closed head injury who had failed medical therapy , including barbiturate coma . cerebellar decompression for mass lesion is a standard neurosurgical response to any process in the posterior fossa ( hemorrhage , tumor , infection , or stroke ) that threatens cerebellar tonsillar herniation . the paper by ruf and colleagues purports to be a pilot study employing decompressive craniectomy in a standardized approach following development of medically refractory icp in the pediatric population . of the six cases presented , three involve bifrontal craniectomies : two unilateral and one cerebellar . the patients underwent surgery between hospital days 1 and 6 and had icp as low as 20 and as high as 70 mmhg . glasgow outcome scale findings are not provided ; however , at least four of the patients appeared to have no more than a mild disability . they randomly assigned 27 patients to craniectomy or medical management alone and found that the craniectomy patients obtained lower icp and better outcomes . in the control group only 14% of children had a favorable outcome , as indicated by 6-month glasgow outcome scale scores . that study suggested ( as statistical significance was not met because of the small numbers ) that aggressive early decompressive craniectomy may benefit this patient group . polin and colleagues explored the use of decompressive bifrontal craniectomy in victims of tbi . this group used a cohort control matching protocol employing the traumatic coma databank to match subjects based on age , icp , radiographic findings , and admission glasgow coma scale score . in a conditional logistic regression analysis comparing all 92 control patients with the craniectomy population , those investigators detected a significant influence of the operation on favorable outcome ( wald = 6.097 ; p = 0.014 ) . medical management alone carried a 3.86-fold greater risk for unfavorable outcome than did decompressive craniectomy . a pediatric subgroup was identified that appeared to benefit from the procedure ( p = 0.025 ) . the authors further identified a subgroup of patients who received surgery within 48 hours and who never had sustained icp elevation over 40 mmhg . the pediatric patients in this subset had favorable outcomes in 8 out of 10 cases , with statistically improved outcomes compared with control individuals . we think it unlikely that decompressive craniectomy will become a commonplace management scheme for tbi . standard medical management allows control of icp while preserving the ability to conduct a neurologic examination . the role of decompression as compared to that of other ' option ' therapies , such as hypothermia and barbiturate coma , is still evolving . the advantage of decompressive craniectomy over these other therapies is the rapid and generally permanent decline in icp , maintenance of neurologic status , and even the ability to obtain a neurologic examination after the procedure is performed . the disadvantages are the need for at least two surgeries ( one to replace the bone flap ) and the theoretical development of bifrontal contusions after decompression . decompressive hemicraniectomy and duraplasty for evacuation of mass lesions and management of unilateral hemispheric swelling is more widely accepted in head injury management . most surgeons have first hand experience with delayed cerebral swelling after removal of a subdural hematoma , presumably caused by venous infarction and/or cerebral contusion . some will remove the bone flap prophylactically after such operations and others the paper by ruf and colleagues provides more ammunition for the argument that decompressive craniectomy is a safe and effective method of icp reduction in severe tbi with associated icp elevation refractory to standard management . however , this approach remains a second tier strategy recommended only at the option level . for this scheme to become more prevalent , a large - scale multicenter trial such as that being planned by coplin and colleagues would be necessary .

first - generation drug - eluting stents have raised concerns regarding the risk of late and very late stent thrombosis compared with bare metal stents and require prolonged dual antiplatelet therapy . despite extensive investigations , the physiopathology of these late events remains incompletely understood . aside from patient- and lesion - related risk factors , stent polymer has been cited as one of the potential causes . in fact , the persistence of durable polymer after complete drug release has been shown to be responsible for local hypersensitivity and inflammatory reactions . third - generation drug - eluting stents with more biocompatible or biodegradable polymers have subsequently been developed to address this problem . in this article , we evaluate and discuss the concept and clinical results ( safety and efficacy ) of a third - generation drug - eluting stent with biodegradable polymer : the nobori stent . more frequently than adults , pediatric victims of severe traumatic brain injury experience diffuse severe cerebral edema without mass lesions . these patients require methods to reduce intracranial pressure quickly and reliably . surgical decompression provides rapid relief of increased intracranial pressure and is an alternative to maximal medical therapy for these individuals . based on previous trials , most of which are anecdotal but now include attempts at case controlled and cohort matched investigations , ruf and colleagues describe a series of six pediatric patients treated with a prospectively implemented protocol of decompressive craniectomy for severe traumatic brain injury . the heterogeneous approaches presented ( which include hemicraniectomy , bifrontal craniectomy , and suboccipital craniectomy ) undermine the applicability of the results . however , this report , coupled with similar papers , does highlight the need for a true controlled trial of this modality to examine whether craniectomy can emerge as more than a second line option for the management of increased intracranial pressure .

a 32-year male patient presented to the ed with severe dyspnea and agitation at 2:00 a.m. the patient was placed in orthopnea position and oxygen was administered . blood pressure was 165/50 mmhg , heart rate was 113 bpm , body temperature was 36.5 c , 25 breaths of respiratory rate per minute and oxygen saturation with pulse oximetry was 78% with nasal cannula . physical examination revealed rales as far as upper zones of both of lungs and at base of heart , and a diastolic grade 2/6 murmur of aortic regurgitation was heard . medical history included hypertension treated with angiotensin receptor blocker ( arb ) and smoking habit . electrocardiography ( ecg ) showed sinus tachycardia with t - wave inversion in leads di and vl . the working diagnosis was pulmonary edema , and use of bronchodilator inhaler was initiated , as well as intravenous nitrates and intravenous diuretic therapy . while taking arterial blood gas sample , patient s condition deteriorated and abdominal respiration pattern became obvious . elective tracheal intubation of patient was performed , and patient was admitted to coronary intensive care unit ( cicu ) with pulmonary edema and possible acute valvular insufficiency . transthoracic echocardiogram ( tte ) showed mild to moderate aortic regurgitation with 3.3 cm of sinus of valsalva . systolic function of ventricle was normal and left ventricular ( lv ) cavity widened to 5.1 cm at end - diastolic volume . arterial blood gas before intubation showed hypoxia with 76% arterial oxygen saturation and hypocarbia with metabolic acidosis of 7.13 ph . contrast - enhanced computed tomography ( cect ) of thorax was used to refine the diagnosis . as a team , the cardiologist , the cardiovascular surgeon , and the radiologist checked the computed tomography ( ct ) images . sinus of valsalva was 3.5 cm and no flap was found in ascending or descending aortas . as lab test results showed coronary ischemia with positive troponin i levels ( 1.016 ng / dl ) , it was decided that a diagnostic coronary angiography ( cag ) would be performed . in the catheterization laboratory , intubation of right coronary artery ( rca ) and left main coronary artery ( lmca ) was difficult , and the test required 1 hour to complete . pulmonary embolism and coronary artery disease as cause of acute dyspnea were ruled out , but the cause of dissection of aorta was still unclear because of possibility of a false negative ct scan . transesophageal echocardiography ( tee ) was selected as the next diagnostic tool due to high pretest probability of aortic dissection . tee showed a stanford type a dissection flap closing lmca ostia from beat to beat and compromising the aortic valve with moderate aortic regurgitation . determining the appropriate diagnosis for acute heart failure was very challenging , but at 6.00 a.m. , surgery to correct a stanford type a dissection localized in the valve and coronary ostia was performed . upon further careful review of ct scan images , a very tiny flap was observed at the ostium of lmca ( figure 1 ) . transverse cut view of contrast enhenced enhanced computed tomography shows a tiny flap at the ostium of lmca that was initially misdiagnosed . acute aortic dissection is a life - threatening medical emergency that can quickly lead to death . incidence is estimated to be 3 in a 1000 cases according to international registry of aortic dissection ( irad ) . if left untreated , 33% of individuals will die within 24 hours of presentation , and 50% die in the initial 48 hours . although diagnostic tests have improved , the condition remains undiagnosed in about half of patients because of variable symptoms and negative laboratory tests . after first admission tests of physical examination , vital signs , and electrocardiogram ( ecg ) ; the most frequently performed tests to diagnose aortic dissection are ct , tee and magnetic resonance imaging ( mri ) . a recent meta - analysis by shiga et al . reviewed published studies of diagnosis of aortic dissection by tee , helical ct and mri showed that these tests have equal and reliable diagnostic value . tee had 99% sensitivity and 95% specificity , helical ct had 100% sensitivity and 98% specificity , and mri had 98% sensitivity and 98% specificity [ 4 , 5 ] . in the present case , first admission physical examination and laboratory tests were supportive of aortic dissection . after a misdiagnosis was made by the radiologist , the cardiologist , and the surgeon based on ct of chest , cag was performed to clarify coronary ischemia , which is contraindicated in aortic dissection . correct diagnosis of aortic dissection was made with tee , and the patient was taken to surgery . further examination of ct scan images then revealed a tiny flap at the ostium of lmca ( figure 1 ) . aortic dissection may occur in a small part of aorta and not be seen in ct scan or laboratory test results . if suspicion of aortic dissection is high , diagnosis can be made with tte and tee in intensive care units ( icus ) . dyspnea is the major symptom of acute heart failure and an etiologic assessment must be made for every patient who presents with acute dyspnea . in the present case , aortic dissection compromised aortic valve and coronary ostium , increasing left ventricle end - diastolic pressure , which led to pulmonary edema and acute dyspnea . in episodes of acute dyspnea though a diagnostic tool like ct scan may have 100% sensitivity and 98% specificity , it may also contribute to a misdiagnosed aortic dissection . if high pretest probability is present and ct scan does not support the diagnosis , tee and mri may be used next to determine the correct diagnosis . debilitating conditions , such as disc herniation or lumbar spinal stenosis , are one of the most common causes of lumbago and lumbar radiculopathy . it may be easily recognized by their clinical symptoms and signs . magnetic resonance imaging ( mri ) nevertheless , there are uncommon causes of lumbago or radiculopathy , which include intraspinal extradural masses , such as synovial cyst , ganglion cyst , pseudocyst , hematoma and metastatic tumor , as well as arachnoid cysts . on the basis of clinical examination and history taking , these uncommon causes are generally difficult to distinguish from those that are more common . of these uncommon causes , intraspinal extradural cysts that communicate with the intervertebral discs are quite rare . this is a case report of a lumbar discal cyst in a patient with low back pain and pain that radiates to the legs . a 40-year - old man disclosed a 6-months history of aggravated lumbago and pain that radiated to the left buttock and the leg . , he showed paravertebral tenderness in the lower lumbar spine with lumbar motion limitation secondary to pain . a neurologic examination was remarkable for a grade 3/5 dorsiflexion weakness in his left ankle . the straight leg raising test on the left was positive at an angle of 45 degrees ( 45 ) . the vas score of lumbago was 4 ( pain was scored on a vas from 0=no pain to 10=strongest pain imaginable ) and that of radiating pain was 8 . an mri scan revealed a disc extrusion at l3/4 and a large spherical lesion behind the l4 vertebral body . a mass lesion revealed a low signal intensity on t1-weighted image and high signal intensity on t2-weighted images , which were located in the left ventromedial aspect of the extradural space at the l4 body level , compressing the thecal sac and left l4 nerve root dorsally ( fig . 1 ) . there was a central disc bulging with a ligamentum flavum thickening at the l4/5 intervertebral disc levels , while a narrowing was detected at the left l4/5 intervertebral foramen . based on further enhanced imaging studies , the cystic mass was well enhanced and seemed to be connected like a stalk between the l3/4 intervertebral disc spaces . this patient unsuccessfully underwent a conservative treatment with rest , nsaids ( non - steroridal anti - inflammatory drugs ) and an epidural steroid injection at a local clinic . owing to worsening of sensory and motor abnormalities in the lower extremities , a left partial laminectomy was performed at l4 ( due to the location of a mass lesion behind the l4 body ) . a darkish red cyst was seen underneath the left l4 nerve root , compressing the thecal sac and displacing the left l4 nerve root dorsally . a meticulous dissection was necessary for the severe adhesions between the cystic mass and nerve root as well as dura mater ( fig . 2 ) . grossly , the cystic mass was round and 1.21.1 cm in size . serous hemorrhagic fluid was aspirated with a 22-gauge syringe and sent for both pathologic study and culture ( fig . histologically , the cyst walls were constituted with dense fibrous connective tissues without cell linings or disc materials . the patient 's radicular pain resolved promptly after surgery and only slight numbness overlying his left anterior thigh was reported during the follow - up observation period of two ( 2 ) years . many articles have been reported about intraspinal extradural masses such as synovial cysts , ganglion cyst , perineural cyst , chordoma and metastatic tumor . among them less than one hundred ( 100 ) case reports and small case series for discal cysts have been published to date1,10 ) . the fact that discal cysts may be present in the absence of additional spinal pathology and most commonly affect young males may be distinctive characteristics that are different from other degenerative conditions . several pathogeneses may be suggested , such as degenerative changes due to previous herniated disc material , hematoma associated with a disc herniation , and mucoid degeneration . kono et al . mentioned that the pathogenesis of discal cysts is similar to that of meniscal cysts of the knee and synovial cysts of the facet joint9 ) . based on this hypothesis , focal degeneration of the intervertebral disc may lead to leakage of fluid into the spinal canal producing an inflammatory reaction . proposed that discal cysts would arise from hemorrhage of an epidural venous plexus due to mechanical forces transmitted by a herniated annular fissure2 ) . suggested that discal cysts were similar to ganglion cysts of the posterior longitudinal ligament or annulus fibrosus12 ) . the clinical symptoms and signs of discal cysts resemble those found in patients with a lumbar disc herniation . plain radiographs have a low diagnostic value except for identifying other conditions , such as degenerative spondylolisthesis and spondylosis . mr imaging demonstrates the nature of the cystic lesion and its relationship to the corresponding disc . like a normal intervertebral disc , a discal cyst reveals a low signal on t1-weighted imaging and a high signal on t2-wighted imaging . in addition , the surrounding rim and contents of a cyst may show enhancement in a contrast enhanced mri . these mr imaging characteristics are the keys to differentiating discal cysts from lumbar disc herniation11 ) . further diagnostic evaluation for a suspected discal cyst may include discography and ct discography13 ) . ct - discography was not incorporated owing to the possibility of aggravating the patient 's motor weakness , which could be provoked by contrast media . moreover , it was possible to confirm the connection between the corresponding disc and cyst intraoperatively . the natural history of the discal cyst is still unknown and therapeutic guidelines have not been established . spontaneous regression of a discal cyst has been reported4,14 ) , as well as regression after intradiscal and epidural steroid injections8 ) . however , the management of a discal cyst is similar to that of a lumbar herniation6 ) . ct - guided percutaneous aspiration can be considered as another initial treatment option for a discal cyst , because it has several potential advantages , including faster recovery , low complication rates and avoidance of general anesthesia3,7,8 ) . surgical resection of a discal cyst is reserved for patients with persistent symptoms . in this case study , this patient 's symptoms totally resolved after surgical excision and he remained nearly asymptomatic postoperatively for two years and thereafter . . they should remain in the differential diagnosis of any intraspinal extradural mass lesion compressing the thecal sac and nerve root . mr imaging helps to confirm the diagnosis , while surgical excision of a cyst should be considered for persistently symptomatic patients .

acute dyspnea is a major complaint of patients admitted to cardiology and emergency departments ( ed ) . acute dyspnea can be life - threatening , and is seen in cases of asthma , pulmonary embolism , acute heart failure and myocardial infarction . the present case is that of a 32-year - old man admitted to the ed with orthopnea position and agitation . physical examination , electrocardiogram ( ecg ) , transthoracic echocardiogram ( tte ) , contrast - enhanced computed tomography ( cect ) of thorax and coronary angiography ( cag ) helped to rule out chest disease pathologies such as pneuomo - thorax , pulmonary embolism and coronary artery disease , but were not enough to make an appropriate diagnosis in this case . because of high pretest probability of aortic dissection , transesophageal echocardiography ( tee ) was performed and a diagnosis of stanford type a dissection closing left main coronary artery ( lmca ) ostia from beat to beat was made . discal cysts are a rare cause of lumbar radiculopathy . there are only a few reports of this disease in medical literature . the authors describe the case of a 40-year - old man with a lumbar discal cyst that led to radiculopathy . an intraspinal extradural cystic mass was responsible for low and high signal intensities observed in lumbar lesions on t1 and t2 weighted magnetic resonance images . this cyst was a grossly spherical mass with clear serous fluid , which was connected to an adjacent intervertebral disc . histopathology of the cystic walls revealed fibrous connective tissues without specific cell linings . clinical symptoms were promptly relieved after surgical resection . further research on the pathophysiology and treatment of discal cysts are needed .

salmonella was the most common cause of foodborne outbreaks ( fbos ) in the european union ( eu ) in 2013 , with 1168 fbos of human salmonellosis reported by 22 member states , accounting for 22.5% of all notified outbreaks of foodborne illness in the eu according to the european food safety authority ( efsa , 2015 ) . the genus salmonella , which is closely related to the genus escherichia , groups gram - negative , non - spore - forming , is rod - shaped bacteria belonging to the enterobacteriaceae family . two species are distinguished : s. enterica , classified into six subspecies ( grimont et al . , 2007 ) , and s. bongori . salmonella enterica subspecies enterica is an intracellular pathogen of warm - blooded mammals comprising over 2500 serovars ( baker and dougan , 2007 ) . some salmonella serovars , such as s. enterica serovar enteritidis and s. enterica serovar typhimurium , are non - specific host pathogens that can colonise a broad range of animals , rarely causing clinical manifestations . in humans , however , they can lead to gastroenteritis or occasionally septicemia ( galanis et al . , the serovars most frequently associated with human illness in the eu are s. enteritidis ( 39.5% ) , s. typhimurium ( 20.2% ) , and monophasic s. typhimurium ( 8.6% ) ( efsa , 2015 ) . salmonella is the most common causative agent , reason for hospitalisation , and cause of death tracked by the foodborne diseases active surveillance network ( foodnet ) ( cdc , 2011 ) . beyond their health effects salmonella alone is responsible for approximately 1 million foodborne infections ( scallan et al . , 2011 ) and it costs 365 million $ in direct medical expenditures annually ( us department of agriculture , 2011 ) . common salmonellosis symptoms include diarrhea , fever , and abdominal cramps beginning 12 to 72 hours after the consumption of contaminated food or beverages ( shariat et al . , 2013 ) . more often , human s. typhimurium cases are associated with the consumption of contaminated pork and beef , whereas s. enteritidis cases are linked to eggs and poultry meat . subclinical infection in animals leads to herd or flock contamination , with intermittent or persistent shedding of bacteria ( efsa , 2013 ) . in laboratory testing for salmonella enterica , isolates are identified and typed by several phenotypic methods , including biochemical profiling , serotyping , and phage typing . standard serotyping methods , based on the detection of somatic ( o ) and flagellar ( h ) antigens , are tedious and time - consuming . although they lack the capacity to fingerprint strains in a sensitive manner , they remain useful in surveillance programmes ( herikstad et al . , 2002 ) . more recently , different genotyping methods have been developed for the genetic discrimination of salmonella isolates in outbreaks , such as multilocus sequence typing , multilocus variable - number tandem - repeat analysis , next generation sequencing . pulsed - field gel electrophoresis ( pfge ) was adopted for salmonella surveillance and outbreak research in the 1990s . because of its remarkable discriminatory power and high reproducibility , it has been successfully used in typing salmonella strains isolated from human patients , foods and feed ( zou et al . , 2013 ) . although pfge is also labour - intensive , public health surveillance laboratories use it to determine strain relatedness and to confirm fbos . indeed , the us cdc recommends pfge as the gold standard method for molecular characterisation in outbreak investigations . in this paper it was reported the investigation of a household / domestic kitchen fbo of salmonellosis occurred in biella on the 15 of january 2010 linked to homemade lasagne . microbiological , serological , and molecular assays on salmonella isolates from human and food samples were performed to determine the relatedness of the implicated salmonella strains , identify the sources of infection , and trace the routes of salmonella contamination in this circumscribed fbo . the study was conducted in collaboration between the food control laboratory at the institute for experimental veterinary medicine of piedmont , liguria and aosta valley , turin , the biella department of public health prevention , and the hospital of biella ospedale degli infermi . four adults ( 1 man and 3 women ; age range , 45 - 61 years ) sought medical attention at the emergency room of the degli infermi hospital , biella because of gastrointestinal symptoms on the 16 of january 2010 . from initial accounts , all four persons were hospitalised , and the local public health and prevention department was notified . a standardised questionnaire was administered by the public health officials in order to obtain demographic and clinical data , history of recent travel , food and water consumption , exposure to animals , and other illness during the week before becoming ill . in the interviews , the patients stated that they had consumed a homemade baked lasagne dish consisting of boiled pasta in which cheese and tomato sauce are mixed and then cooked in the oven . then , a mixture of raw eggs was added to homemade lasagne and left in switched off oven until the evening . before eating in addition , the patients had also eaten salad , turkey meat , and cake . people who consumed common meal on the 15 of january 2010 in biella , who had gastro enteric symptoms from 15 to 16 of january , had not gastro enteric symptoms before the 15 of january 2010 . the food sample was analysed to identify salmonella spp . ; the stool samples were tested for salmonella , shighella and campylobacter . salmonella from the food sample was isolated according to iso 6579:2002/cor 1:2004 ( iso , 2004 ) . this method entails several steps : pre - enrichment in non - selective liquid medium ( buffered peptone water ; biokar diagnostic , beauvais cedex , france ) ; enrichment in two selective liquid media [ rappaport - vassiliadis medium with soya broth ( microbiol diagnostici , cagliari , italy ) and muller - kauffmann tetrathionate novobiocin mkttn broth ( liofilchem srl , roseto , te , italy ) ] ; and plating on xylose lysine deoxycholate agar ( xld ) ( microbiol diagnostici ) and brilliant green agar ( bga ) ( microbiol diagnostici ) . an internal method for detecting salmonella , shighella and campylobacter specifically , for the isolation of salmonella and shighella have been used hektoen enteric agar ( oxoid , rodano , mi , italy ) , mac conkey agar ( oxoid ) and salmonella shighella agar ( oxoid ) ; for the isolation of campylobacter the stool samples were analyzed using campylobacter species ag rapid test ( li starfish s.r.l . , cernusco , mi , italy ) and campylobacter selective agar ( biomrieux , marcy letoile , france ) . all isolated strains were identified by conventional biochemical methods ( api 20e ; biomriux ) and the salmonella spp . were serotyped according to the kauffman - white - le minor classification scheme ( kauffmann , 1966 ; le minor and popoff , 1987 , grimont and weill , 2007 ) . the isolates were processed according to the pfge protocol described by the us centers for disease control and prevention ( cdc , 2013 ) using xbai ( carlo erba reagents srl , cornaredo , mi , italy ) and blni ( roche diagnostics corporation , indianapolis , in , usa ) restriction enzymes . pfge using a single restriction enzyme ( xbai ) is a standard method for genotyping s. enteritidis ( dewaele et al . , 2012 ) . thus , the ability to deduce the serotype of a salmonella isolate based on its pfge profile provides an alternative method for screening and identifying salmonella serotypes ( zou et al . , 2010 ) . the pfge fingerprint patterns were analysed with bionumerics software ( version 7.1 ; applied maths , sint - martins - latem , belgium ) and the pfge patterns were normalised by interpolation to the nearest reference lane ( salmonella braenderup strain atcc h9812 ) . for comparing the pfge profiles , values of optimization of 1.0% and position tolerance of 1.0% were applied to both enzymes . dice similarity coefficients were calculated on the basis of pairwise comparisons of the pfge profiles . four adults ( 1 man and 3 women ; age range , 45 - 61 years ) sought medical attention at the emergency room of the degli infermi hospital , biella because of gastrointestinal symptoms on the 16 of january 2010 . from initial accounts , all four persons were hospitalised , and the local public health and prevention department was notified . a standardised questionnaire was administered by the public health officials in order to obtain demographic and clinical data , history of recent travel , food and water consumption , exposure to animals , and other illness during the week before becoming ill . in the interviews , the patients stated that they had consumed a homemade baked lasagne dish consisting of boiled pasta in which cheese and tomato sauce are mixed and then cooked in the oven . then , a mixture of raw eggs was added to homemade lasagne and left in switched off oven until the evening . before eating in addition , the patients had also eaten salad , turkey meat , and cake . people who consumed common meal on the 15 of january 2010 in biella , who had gastro enteric symptoms from 15 to 16 of january , had not gastro enteric symptoms before the 15 of january 2010 . stool samples were collected from all patients . only residues of the baked lasagne could be collected for sampling and subsequent analysis . the food sample was analysed to identify salmonella spp . ; the stool samples were tested for salmonella , shighella and campylobacter . salmonella from the food sample was isolated according to iso 6579:2002/cor 1:2004 ( iso , 2004 ) . this method entails several steps : pre - enrichment in non - selective liquid medium ( buffered peptone water ; biokar diagnostic , beauvais cedex , france ) ; enrichment in two selective liquid media [ rappaport - vassiliadis medium with soya broth ( microbiol diagnostici , cagliari , italy ) and muller - kauffmann tetrathionate novobiocin mkttn broth ( liofilchem srl , roseto , te , italy ) ] ; and plating on xylose lysine deoxycholate agar ( xld ) ( microbiol diagnostici ) and brilliant green agar ( bga ) ( microbiol diagnostici ) . an internal method for detecting salmonella , shighella and campylobacter was carried out on the stool samples . specifically , for the isolation of salmonella and shighella have been used hektoen enteric agar ( oxoid , rodano , mi , italy ) , mac conkey agar ( oxoid ) and salmonella shighella agar ( oxoid ) ; for the isolation of campylobacter the stool samples were analyzed using campylobacter species ag rapid test ( li starfish s.r.l . , cernusco , mi , italy ) and campylobacter selective agar ( biomrieux , marcy letoile , france ) . all isolated strains were identified by conventional biochemical methods ( api 20e ; biomriux ) and the salmonella spp . were serotyped according to the kauffman - white - le minor classification scheme ( kauffmann , 1966 ; le minor and popoff , 1987 , grimont and weill , 2007 ) . the isolates were processed according to the pfge protocol described by the us centers for disease control and prevention ( cdc , 2013 ) using xbai ( carlo erba reagents srl , cornaredo , mi , italy ) and blni ( roche diagnostics corporation , indianapolis , in , usa ) restriction enzymes . pfge using a single restriction enzyme ( xbai ) is a standard method for genotyping s. enteritidis ( dewaele et al . , 2012 ) . thus , the ability to deduce the serotype of a salmonella isolate based on its pfge profile provides an alternative method for screening and identifying salmonella serotypes ( zou et al . , 2010 ) . the pfge fingerprint patterns were analysed with bionumerics software ( version 7.1 ; applied maths , sint - martins - latem , belgium ) and the pfge patterns were normalised by interpolation to the nearest reference lane ( salmonella braenderup strain atcc h9812 ) . for comparing the pfge profiles , values of optimization of 1.0% and position tolerance of 1.0% dice similarity coefficients were calculated on the basis of pairwise comparisons of the pfge profiles . epidemiological investigation revealed the main symptoms of nausea ( 50% ) , abdominal cramps ( 75% ) , diarrhea ( 100% ) , vomiting ( 100% ) , fever ( 100% ) , and low back pain ( 25% ) . the symptoms began 5 hours after eating the meal in the first case and 9 hours later in the last case . during the interview no history of recent travel , food and water consumption , exposure to animals , and other illness during the week before becoming ill was reported . a participant cooked the baked lasagne at the dinner and all the people ate the lasagne . was isolated in three patients ( stool samples ) and in the baked lasagne ( residues stored in the refrigerator ) ; one patient was tested negative for all pathogens . four s. enteritidis isolates were genotyped by pfge using xbai and blni restriction enzymes , which yielded consistent reproducible fingerprints . single enzyme restriction analyses by xbai distributed the four isolates into one cluster ( figure 1 ) . s. enteritidis is the major serovar associated with human salmonellosis linked to the consumption of contaminated poultry products , including eggs ( braden , 2006 ; much et al . , 2009 ) . in 2013 207 strong evidence outbreaks were attributed to the consumption of eggs and egg products and 59.9% of fbos were caused by s. enteritidis ( efsa , 2015 ) . a 2002 - 2003 us case - control study reported that a significant risk factor of salmonella infections ( or 2.7 , 95% ci 1.1 - 3.9 ) is consuming undercooked eggs or egg - containing dishes inside the home ( middleton et al . , 2014 ) . the household / domestic kitchen remains the most commonly reported setting in european fbos ( 38.5% ) followed by the public / commercial kitchens ( restaurant , caf , pub , bar , hotel categories ) in 2013 ( 22.2% ) ( efsa , 2015 ) . the domestic fbo outbreak described here was linked to the homemade lasagne contaminated with salmonella spp . we identified a common source of infection by using pfge , one of the most important molecular characterization methods of diagnostic testing to compare bacteria strains . how and at which point during food preparation the lasagne became infected with salmonella can not be definitively determined , but the data suggest opportunities for person to food , food to food , and equipment to food cross - contamination . generating a hypothesis about the likely sources , the most probable is that the eggs used in preparing the lasagne were probably contaminated with salmonella enteritidis and their adding after cooking process is responsible of the contamination of rte product . indeed the reheating process before consumption normally applied at low temperature is not enough to kill the bacteria and might even to promote the bacterial growth . a plausible alternative hypothesis is that the lasagne was contaminated with salmonella enteritidis after cooking due to cross - contamination . indeed , one of the most common causes of fbos is cross - contamination during food handling due to the use of improperly washed utensils and handling by ill or healthy carriers . serotyping according to the kauffman - white - le minor classification scheme and pfge analysis allowed identifying the source of food poisoning in this domestic fbo . however , it was arduous to single out which ingredient of the meal was the source of salmonella contamination . collaborative efforts by the hospital physicians , public health department officials , and laboratories using different laboratory analysis methods during the outbreak investigation were essential for identifying the causative agent . good sanitation , safe food handling and preparation practices in the entire food chain are key to preventing foodborne illness . consumers should be made aware of the risks of fbo and learn how to minimise their chances of becoming ill by considering food safety at each step , from food purchase , to cooking , cleaning , and storing leftovers appropriately ( international food information council foundation , 2014 ) . peripheral nerve injuries are commonly caused by trauma to the upper limbs and are present in an estimated 23% of all patients admitted to a level 1 trauma center ( noble et al . , 1998 ) . the economic impact of nerve injuries can be large with operative costs , hospital charges , rehabilitation visits , and lost time at work . only subtle improvements to peripheral nerve repair have been made recently , and our current knowledge of nerve physiology and regeneration vastly exceeds our current repair capabilities . poor outcomes of peripheral nerve injury are largely due to the slow process of axonal outgrowth . after nerve injury , severed proximal axons with intact cell bodies can grow up to 1mm / day . regenerating axons preferentially target appropriate end organ receptors but do not always take a direct route to their target ( fox et al . , 2012 ) . thus , functional recovery is not always obtained due to slow growth and poor alignment of motor and sensory fibers . muscle atrophy initiates immediately after muscle denervation , and if motor axons do not reach their target muscle within a critical time window , muscle tissue is less receptive to re - innervation . the most common current strategies to augment recovery after nerve injury such as decellularized nerve allografts , tissue matrices and nerve growth guides involve techniques to enhance axonal regeneration and decrease surrounding inflammation . despite these advancements , the combination of slow axonal outgrowth , wallerian degeneration , and muscle atrophy are still barriers to significant progress . invertebrate axonal fusion after nerve injury is a natural mechanism that promotes rapid and highly specific recovery of neurons . given the mechanism of axonal fusion is possible , peg has been investigated as an agent to promote axonal fusion in vertebrates . bittner et al . pioneered initial studies using peg to fuse crayfish and giant earthworms axons with improvement in axonal fusion when axonal endings were exposed to a calcium free saline solution ( bittner et al . the addition of an anti - oxidant such as methylene blue ( mb ) was noted to have an additional positive impact in rat sciatic nerve repair ( spaeth et al . , the proposed mechanism is peg - induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur . in an axonal injury without peg , axonal endings seal after an influx of calcium , preventing axonal fusion ( yoo et al . , 2003 ) . figure 1 demonstrates the bioengineered process of peg fusion . when severed axonal endings are exposed to calcium - free hypotonic saline and an antioxidant ( i.e. , methylene blue or melatonin ) , vesicle - mediated sealing peg is then applied to artificially induce closely apposed membranes of severed axonal ends to flow into each other and fuse . this produces a partial repair of the plasmalemmal membranes that are then perfused with calcium containing saline , which causes vesicles to accumulate and seal remaining holes at the injury site . mechanism of polyethylene glycol ( peg ) axonal fusion . reprinted from rodriguez - feo et al . the success of the peg - fusion technique is based on multiple steps and factors . peg , a hydrophilic compound , enhances axonal fusion in either severed or crushed settings and restores the ability to generate compound action potentials across the site of injury . peg potentially facilitates lipid bilayer fusion by removing water molecules from the lipid bilayer at or near the damage site ( figure 1 ) . sealing of axolemmal damage occurs through a calcium - dependent accumulation of membranous structures that interact with nearby undamaged membrane to form a plug . in severed nerves , this calcium - dependent system plugs the cut ends of axons preventing them from fusing with an adjacent axonal stump . our studies have taken advantage of this calcium - dependent process by incubating in a calcium - free solution prior to nerve fusion , which prevents axolemmal sealing thereby enhancing peg based fusion ( sexton et al . oxidative damage has also been shown to play a role in ischemia reperfusion injury after peripheral nerve injury . methylene blue has been previously shown to slow axolemmal sealing and thereby enhance behavioral recovery after nerve injury ( spaeth et al . , methylene blue has shown a protective effect if applied in the correct sequence ( i.e. , after nerve severance and before peg based fusion ) . taken together , these studies have shown that peg , with carefully controlled modification of the periaxonal environment can fuse simple nerve transections . we have used the fusogenic properties of peg , the advantages of a calcium - free irrigation solution , and the antioxidant properties of methylene blue to demonstrate a rapid and decisive recovery of completely severed sciatic nerves in a commonly accepted mammalian model of peripheral nerve injury . most current research efforts have focused on investigating peg fusion in a rat sciatic nerve model . the rat sciatic nerve is easily accessible through a 23 cm incision posterior to the femur . after a small cutdown to the nerve , the perineural tissue can be dissected easily to free the nerve for transection and repair . our group has demonstrated the efficacy of peg fusion after neurotmesis with direct repair , nerve autografts , and nerve allografts . compound action potentials ( cap ) are measurements of nerve conductivity and can be readily measured across healthy nerves . after nerve transection , caps can no longer be obtained due to axonal discontinuity . caps are not restored after a direct suture repairdue to lack of axonal fusion secondary to axolemmal sealing after injury . however , after peg axonal fusion , caps are restored . bittner et al . published cap data after direct suture repair with and without peg ( bittner et al . , 2012 ) . baseline caps were obtained in all rats prior to nerve injury ( mean 4.1 0.16 mv ) . postoperatively , caps were not measurable immediately after repair in the control group ( direct suture repair ) . however , caps were restored within minutes after repair ( mean 3.3 0.23 mv ) in the peg fusion group . , baseline caps were obtained in all rats prior to nerve injury ( mean 4.131 2.25 mv , n = 20 ) . postoperatively , caps were not measurable immediately after repair or after 72 hours in the control group ( direct suture repair ) . however , caps were restored immediately after repair ( mean 3.533 1.61 mv , n = 10 ) and after 72 hours ( mean 2.051 0.857 mv , n = 10 ) in the peg fusion group . our experience with cap restoration in the peg fusion group was similar when using peg fused allografts ( riley et al . , 2015 ) . our rat behavioral testing includes both the sciatic function index ( sfi ) and foot fault asymmetry score ( ff ) . behavioral recovery was superior in the peg fused group in direct repair at 12 weeks ( bittner et al . , 2012 ) as well as the peg fused group in nerve allografts at 6 weeks ( riley et al . our nerve autograft study evaluated rats up to 3 days , and the peg fused group already had superior behavioral outcomes ( sexton et al . , 2012 ) . figure 2 demonstrates long - term behavioral outcomes after a standard microsurgical repair compared to a peg fusion repair after nerve transection . when considering the electrophysiology and behavioral data together , it suggests at least a partial inhibition of wallerian degeneration after peg fusion . behavioral assessments of sciatic nerve function according to the sciatic functional index ( a ) and foot - fault asymmetry test ( b ) . sciatic function index ( sfi ) and foot fault asymmetry score ( ff ) scores ( mean se ) were assessed from 3 days to 12 weeks post - operatively for two groups : control ( black dotted line ) and polyethylene glycol ( peg ) ( light - gray dotted line ) . individual animals for all 12 weeks animals were graphed according to the aforementioned group color scheme ( data not yet published , manuscript in preparation ) . after axonal injury , motor and sensory nerve counts are decreased distal to the injury site . immunohistochemical staining using commercial antibodies against carbonic anhydrase ii ( ca2 ) and choline acetyletransferase is a method to stain for sensory and motor neurons respectively . our research has shown increased sensory and motor axon counts in peg - treated nerve repairs ( figure 3 ) . toluidine blue staining of peg fused nerves shows higher proportions of healthy myelinated axons compared to negative control nerves . additionally , our lab has demonstrated the expression of npy1 , a marker of wallerian degeneration , in negative control nerves , whereas this is not expressed in peg - fused nerves . these findings imply that we have delayed or even inhibited wallerian degeneration by fusing axons and maintained both axonal viability and function . control ( a c ) and polyethylene glycol ( peg ) ( d f ) stained for choline acetyltransferase to label motor axons at 1 week ( a , d ) , 4 weeks ( b , e ) , and 12 weeks ( c , f ) post - operatively ( data not yet published , manuscript in preparation ) . diffusion tensor imaging ( dti ) is a magnetic resonance imaging ( mri ) technique , which is an emerging diagnostic tool in nerve injury . the institute of imaging science at our institution has developed a novel imaging protocol using dti for peripheral nerves . utilizing this protocol , we have been able to demonstrate the restoration of axonal continuity after peg fusion repair ( figure 4 ) . representative tractography images show that axonal tracts do not extend more than a couple millimeters distal to the repair site in control nerves ( cut + suture , n = 6 ) . in peg fused nerves ( n = 6 ) , the peg group had a statistically significant increase in the number of tracts that traveled through the repair site ( data not published , manuscript in progress ) . our mri data supports the interpretation that peg - fused nerves establish axonal fusion which has been previously evaluated through behavioral testing and axon counts . representative tractography of transected nerves harvested immediately following repair : control ( left ) and polyethylene glycol ( peg ) ( right ) . green arrows indicate zone of repair ( data supplied by riley dc and kelm nd , data not yet published , manuscript in preparation ) . despite the effectiveness we have observed with peg fusion , there can be inconsistencies in morphological and functional recovery of peg - fused animals . as demonstrated in figure 4 , peg fusion has not fully restored dti characteristics in these nerves , perhaps suggesting that not all axons were fused . we speculate that a variability in the number of fused axons may in turn be responsible for the variabilities in morphological and functional recovery . we also suspect factors such as the quality of repair , time of surgical intervention , and a surgeon 's level of experience with the technique play a critical role in achieving successful peg fusion . our studies have included applying the peg solution manually through a syringe with the needle held at the surface of the neurorrhaphy . this method , potentially , could result in small movements of the needle resulting in sub - optimal delivery of the peg solution or disturbance of the neurorrhaphy . our current efforts in developing a device are focused on achieving a consistent nerve repair with uniform peg application to the neurorrhaphy site . we recently described our experience with peg fusion using a nerve tube , which helps eliminate the need for sutures at the repair site ( sexton et al . , 2015 ) . our ultimate goal is to develop a simple coaptation system that will allow acute repair of severed nerves , with or without segmental injury , with minimal microsurgical training . our lab has focused its efforts on small animal models , and the study of peg fusion has occurred across multiple laboratories and animal models . we have demonstrated repeated success in working in a rat sciatic nerve model . bittner et al . has demonstrated the efficacy of peg fusion in invertebrate and small animal models in the aforementioned studies ( bittner et al . , 1986 ; in addition , peg fusion has been investigated in large animal sciatic nerve and ex - vivo central nerve models in other laboratories demonstrating reproducibility of the technique ( donaldson et al . , 2002 ; looking forward , we are currently conducting a phase i clinical trial of peg fusion in human nerve repair , and we were also hoping to move towards more large animal studies in the near future . the ability to fuse severed nerves and regain rapid functional recovery could result in a paradigm shift in all aspects of patient care following peripheral nerve damage . because this technique involves the use of current microsurgical techniques , with the simple addition of peg , it could rapidly be incorporated into a neurorrhaphy and autografting regimen . our current clinical trial will investigate efficacy and safety of peg fusion in humans , and our development of a peg delivery device will make peg fusion reproducible with minimal training required to achieve superior results .

in the latest year , and also in 2013 , salmonella was the most frequently detected causative agent in foodborne outbreaks ( fbos ) reported in europe . as indicated in efsa report ( 2015 ) the serotypes mostly associated to fbos are s. typhimurium and enteritidis ; while salmonella typhimurium is generally associated with the consumption of contaminated pork and beef , fbos due to salmonella enteritidis are linked to eggs and poultry meat . in this study it is described the investigation of a domestic fbo involving four adults and linked to homemade lasagne . investigations were performed to determine the relatedness of salmonella strains , identify the sources of infection , and trace the routes of salmonella contamination in this fbo . salmonella strains were isolated in 3 out of 4 patient stool samples and from lasagne and all of them were serotyped as s. enteritidis . pulsed - field gel electrophoresis ( pfge ) analysis revealed the genotypical similarity of all the strains . although serotyping and pfge analysis identified the common food source of infection in this fbo , it was not possible to determine how or at what point during food preparation the lasagne became contaminated with salmonella . the management of traumatic peripheral nerve injury remains a considerable concern for clinicians . with minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury , outcomes of surgical intervention have been unpredictable . the inability to manipulate the pathophysiology of nerve injury ( i.e. , wallerian degeneration ) has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration ( ~1 mm / day ) . when axons are severed , the endings undergo calcium - mediated plasmalemmal sealing , which limits the ability of the axon to be primarily repaired . polythethylene glycol ( peg ) in combination with a bioengineered process overcomes the inability to fuse axons . the mechanism for peg axonal fusion is not clearly understood , but multiple studies have shown that a providing a calcium - free environment is essential to the process known as peg fusion . the proposed mechanism is peg - induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur . this review highlights peg fusion , its past and current studies , and future directions in peg fusion .

synaptic plasticity or long - lasting alterations in the efficacy of synaptic connections between two neurons has been proposed to be the cellular substrate of learning and memory ( malinow and malenka , 2002 ; bredt and nicoll , 2003 ; collingridge et al . , 2004 ; neves et al . , 2008 ) . persistent changes in synaptic efficacy are thought to involve at least two distinct phases an early phase ( on the order of minutes to hours ) that is independent of new protein synthesis and a more long - lasting late phase ( on the order of hours to days ) that is dependent upon new protein synthesis ( bramham and wells , 2007 ; bramham , 2008 ; richter and klann , 2009 ) . the first mechanism typically involves activity - dependent alterations in the phosphorylation and number of postsynaptic alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors ( ampa receptor ) and n - methyl d - aspartate receptors ( nmda receptor ) , which are delivered to the postsynaptic membrane via exocytosis and removed via endocytosis ( collingridge et al . , 2004 ; lau and zukin , 2007 ; neves et al . , 2008 ; holtmaat et al . , 2009 ; newpher and ehlers , 2009 ) . the second more long - lasting mechanism , postulated to underlie long - lasting memory storage , involves local or on - site protein synthesis in dendrites by translational machinery strategically localized in and around synaptic sites ( bramham and wells , 2007 ; bramham , 2008 ; richter and klann , 2009 ) . the identities of the newly synthesized proteins that are required to maintain altered synaptic efficacy are , however , as yet unclear . considerable evidence indicates that alterations in synaptic strength are locked - in by alterations in structural remodeling . these morphological changes include induction of new dendritic spines , enlargement of spines already extant , and the splitting of single spines into two or more functional synapses ( bourne and harris , 2008 ; holtmaat et al . more recently , studies involving spine long - term potentiation ( ltp ) , in which single spines are activated via photolysis of caged glutamate and imaged by means of two - photon microscopy , demonstrate that ltp is accompanied by enlargement of dendritic spines ( matsuzaki et al . , 2004 ; harvey and svoboda , 2007 ; harvey et al . , 2008 ; lee et al . , 2009 ) . mrna translation , a complex process involving the participation of many proteins and rnas , is generally divided into three phases : initiation , elongation , and termination . although each of these steps serves as a point of regulation to control the amount of protein that is produced , initiation is by far the most important and the salient features of this segment are important to keep in mind when considering how synapse stimulation induces changes in the translational apparatus . initiation begins with the formation of a ternary complex comprised of gtp , met - trna , and the initiation factor eif2 ( pestova et al . , 2007 ) . together with additional initiation factors ( eif3 , eif5 , eif1 , and eif1a ) , the ternary complex associates with the 40s ribosomal subunit to form a 43s pre - initiation complex ( chaudhuri et al . , 1999 ; majumdar et al . , 2003 ) . in mammals , this large bolus of factors is recruited to the 5 end of the mrna through interactions with the initiation factors eif4 g and eif3 ; at this point , it is referred to as the 48s pre - initiation complex ( lamphear et al . , 1995 ) . the dexh box protein dhx29 , a putative helicase , is also a required component of this complex for the translation of mrnas with highly structured 5 utrs ( pisareva et al . , 2008 ) . proper positioning of the complex on the 5 end requires the mg cap - binding complex consisting of eif4e , eif4 g and eif4a , which together are called eif4f ( gingras et al . , 1999 ) . with the aid of the initiation factors eif1 , eif1a , and dhx29 , the 48s complex then scans the mrna in the 3 direction until it encounters an aug initiation codon in the correct context , generally the first aug , where it is joined by the 60s subunit to begin polypeptide elongation ( pestova et al . , 1998 ; pestova and kolupaeva , 2002 ; pisareva et al . , 2008 the association between eif4e , the cap binding factor , and eif4 g , is often regulated upon synaptic activity . the protein 4e - bp can bind eif4e and prevent eif4 g from joining with eif4e , thus abrogating cap - dependent translation . phosphorylation of 4e - bp either directly or indirectly by the kinase mammalian target of rapamycin ( mtor ) causes it to dissociate from eif4e , thereby liberating the factor to bind both the cap and eif4 g and initiate translation ( gingras et al . , 1999 ) . such localized stimuli include those that trigger synapse formation , pruning , neurite outgrowth / collapse , activity - dependent synaptic plasticity and injury - induced axonal regeneration . a critical regulator of activity - dependent protein synthesis in dendrites is metabotropic glutamate receptors ( mglurs ) . mglurs are g protein - coupled receptors enriched at excitatory synapses throughout the brain where they act to regulate glutamatergic neurotransmission ( nakanishi , 1994 ; pin and duvoisin , 1995 ; conn and pin , 1997 ) . group i mglurs ( mglur1/5 ) activate phospholipase c , leading to mobilization , and the extracellular signal - regulated kinase ( erk)mitogen - activated protein kinase ( mapk ) pathway through which they modulate signal - to - nucleus communication . signaling by mglur1/5 is critical to synaptic circuitry formation during development and is implicated in forms of plasticity including ltp ( aiba et al . , 1994 ) , long - term depression ( ltd ) ( aiba et al . , 1994 ; oliet et al . , 1997 ; palmer et al . , 1997 ; huber et al . , 2001 ; bear et al . , 2004 ) associative learning ( aiba et al . , 1994 ) and cocaine addiction ( kenny and markou , 2004 ) . mglur1/5 elicit synapse - specific modifications in synaptic strength and spine morphology by stimulating rapid local translation of dendritic mrnas including fmr1 , encoding fragile x mental retardation protein ( fmrp ) ( greenough et al . , 2001 ) . expression of mglur - ltd at schaffer collateral to ca1 pyramidal cell synapses is mediated by persistent internalization of ampars and in adolescent wild - type ( wt ) mice requires de novo protein synthesis ( huber et al . , 2000 ; snyder et al . , 2001 ) that is dependent on the mtor ( hou and klann , 2004 ) . the protein synthesis dependent is both age and region - specific in that ca1 mglur - ltd in neonates ( nosyreva and huber , 2005 ) and mglur - ltd at cortical synapses ( desai et al . , 2006 ) mammalian target of rapamycin is a central regulator of cell growth and proliferation , autophagy , ribosome biogenesis , and translation ( hay and sonenberg , 2004 ; klann and dever , 2004 ; sarbassov et al . , 2005 ) and a critical signaling pathway downstream of group i mglurs . in neurons , components of the mtor signaling cascade are present at synapses and are thought to influence synaptic plasticity via regulation of local protein synthesis ( tang and schuman , 2002 ) . mtor is activated in dendrites by stimulation of group i mglurs ( klann and dever , 2004 ) and is required for mglur - ltd at schaffer - collateral to ca1 synapses ( hou and klann , 2004 ; banko et al . , 2006 ; antion et al . , growing evidence indicates that dysregulation of mtor is associated with human diseases , including cancer , diabetes , and autism ( jaworski and sheng , 2006 ; sabatini , 2006 ; dann et al . , 2007 ) . the fragile x syndrome ( fxs ) is the most common heritable form of mental retardation ( chiurazzi et al . , 2003 ; jacquemont et al . , 2007 ; bassell and warren , 2008 ) as well as the most common known genetic cause of autism ( fryns et al . , 1984 ) . fxs is caused by loss - of - function mutations in the fmrp , which is encoded by the fmr1 gene located on the x chromosome . in humans , the fxs typically results from expansion of a cgg repeat sequence in the 5-untranslated region and silencing of the fmr1 gene ( feng et al . , 1995 ; patients with the fxs exhibit a wide - range of neurological deficits including cognitive impairment , seizures , emotional instability , sleep disorders , attention deficits , autonomic dysfunction , and autism ( hagerman and hagerman , 2002 ; o'donnell and warren , 2002 ; chiurazzi et al . , 2003 ; jin and warren , 2003 ) . fmrp , the gene product of the fmr1 gene , is an rna binding protein that associates with many mrnas , some of which encode proteins important for neuronal development and plasticity . fmrp controls activity - dependent dendritic mrna localization ( dictenberg et al . , 2008 ) , stability ( brown et al . , 2001 ; miyashiro et al . , 2003 ; d'hulst et al . 2007 ) and translational efficiency of dendritic mrnas in response to stimulation of mglurs ( weiler et al . , 1997 ; todd et al . , 2003 ; muddashetty et al . , 2007 ; westmark and malter , 2007 ; napoli et al . , 2008 ) . fmrp is detected in cell bodies , in dendritic shafts and branch points ( ferrari et al . , 2007 ) , and at the base of synaptic spines and spine heads ( feng et al . , 1997 ) . fmrp mrna is translated near synapses in response to neurotransmitter activation ( weiler et al . , 1997 ) ; moreover , different models of experience - dependent plasticity such as whisker stimulation ( todd and mack , 2000 ) , visual experience ( gabel et al . , 2004 ) and exposure to enriched environment ( irwin et al . , 2005 ) also promote fmrp mrna translation . exhibit abnormalities in dendritic spine morphology ( bagni and greenough , 2005 ) , cognitive deficits ( o'donnell and warren , 2002 ) , exaggerated , protein synthesis - dependent ltd at schaffer collateral to ca1 pyramidal cell synapses in adolescent wt mice , but protein synthesis - independent in neonatal wt mice ( nosyreva and huber , 2005 ) and in fmr1 ko mice ( huber et al . , 2002 ; koekkoek et al . , 2005 ; hou et al . , 2006 ; nosyreva and huber , 2006 ; zhang et al . , 2009 ) , and decreased ltp in the cortex ( li et al . , 2002 recent findings indicate that dysregulation of mtor signaling in the hippocampus is an important functional link between overactivated mglur signaling , aberrant protein synthesis , and exaggerated mglur - ltd in fmr1 ko mice ( sharma et al . , 2009 ) . for example , the fmr1 ko mouse exhibits aberrant numbers and/or activity of mglur5 ( giuffrida et al . , 2005 ; dolen et al . , 2007 ) , nmda ( gabel et al . , 2004 ; desai et al . , 2006 ) and ampa ( pilpel et al . , 2009 ; schuett et al . , a hallmark feature of the fxs in humans , also observed in the fmr1 ko mouse , is that of synaptic spine dysmorphogenesis . spines on hippocampal and cortical neurons of fmr1 ko mice exhibit enhanced density and are thinner and longer than those of age - matched wt mice , resembling an immature morphology . these observations indicate that fmrp normally controls processes involved in synapse maturation , stabilization , and elimination ( bagni and greenough , 2005 ) . electrophysiology experiments using cultured neurons indicate a delay in establishing synaptic connections during a brief window in brain development ( braun and segal , 2000 ) . in the somatosensory cortex , although spine dysmorphogenesis persists into adulthood ( galvez and greenough , 2005 ; restivo et al . , 2005 ) , it can be rescued if the animals ( mice ) are exposed to enriched environmental conditions ( restivo et al . , 2005 ; meredith et al . , 2007 ) synaptic activity in the brain can trigger long - lasting changes in synaptic strength ; i.e. , ltp and ltd . early in development , ltp seems to be important for retaining nascent synapses , whereas ltd is more directly involved in activity - guided synapse elimination . the combination of these two phenomena contributes to the essential brain function of learning and memory . impaired synaptic activity almost certainly results in impaired mental development ( vaillend et al . , 2008 ) . new proteins that contribute to re - arrangements and re - assembling of synapse are made locally from mrnas that are trafficked to dendrites ( steward and levy , 1982 ) . local changes in synaptic efficacy are protein synthesis dependent ( steward and schuman , 2003 ; martin and zukin , 2006 ; lin and holt , 2008 ) ; synaptic activity also promotes an increase in the number of polysomes at spines , presumably reflecting increased on - site local synthesis of proteins critical to alterations in synaptic efficacy and structural remodeling of spines ( ostroff et al . , 2002 ) . in neurons , fmrp is packaged in ribonucleoprotein ( rnp ) granules , some components of which have been identified ( ohashi et al . , 2002 ; brendel et al . ; dictenberg et al . , 2008 ; napoli et al . , 2008 ) . fmrp granules contain several proteins involved in translation ( pur alpha and beta , hnrnpu , staufen , ef1alpha , papb1 , eif4e , ribosomal proteins ) , cytoskeleton dynamics ( tubulin , tau , actin , rac1 , cyfip ) , and the movement of molecules and organelles ( dynein , myosins and kinesins ) . importantly , camkii and arc , targets of fmrp that are critical to synaptic plasticity and known to be synthesized de novo in response to neuronal activity , are detected in these granules ( kanai et al . , 2004 ) . fmrp - containing rnp granules are transported along microtubules in dendrites ( antar et al . , 2004 ; ferrari et al . , 2007 these data strongly support a role for fmrp in dendritic delivery of rna and regulation of local translation in specific synaptic compartments . the molecular mechanism(s ) by which fmrp controls translation has been investigated by assessing the polysome / mrnp distribution of the fmrp - containing complex . initially , fmrp was shown to co - sediment entirely with polyribosomes or even larger granules ( ohashi et al . 2004 ; stefani et al . , 2004 ) or to co - sediment essentially equally with polysomes and mrnps ( feng et al . , 1997 ; brown et al . , 2001 ; ohashi et al . , 2002 ) . other laboratories found that fmrp co - sedimented mainly with the mrnp fraction ( siomi et al . , 1996 ; ishizuka et al . , 2002 ; zalfa et al . , 2003 ; while this apparent discrepancy could arise owing to differences in methodologies , brain region or cell type , a recent study by bagni and colleagues shows that fmrp regulates synaptic translation at the level of initiation ( napoli et al . , 2008 ) . these authors showed that fmrp inhibits translation initiation through an interacting factor , cyfip1/sra1 , which binds the cap binding factor eif4e . interestingly , cyfip1 binds eif4e via a novel domain with a characteristic reverse l shaped structure that is also assumed by the canonical eif4e - binding motif ( marcotrigiano et al . , 1999 ) . the importance of cyfip1 in the fmrp - regulatory circuit was demonstrated by an increased level of proteins encoded by known fmrp target mrnas upon reduction of cyfip1 in neurons . furthermore , the brain cytoplasmic rna 1 , another fmrp binding partner , increases the affinity of fmrp for the cyfip1-eif4e complex in the brain ( napoli et al . , 2008 ) . fmrp has been reported to sediment in both the rnp fractions ( i.e. , less the 80s monosome ) as well as with polysomes in sucrose gradients . in the rnp fractions , , we show that cyfip1 inhibits translational initiation by binding initiation factor eif4e and preventing assembly of eif4e with eif4 g . in the polysome fractions , fmrp may inhibit protein synthesis by inhibiting polypeptide elongation ( i.e. , by retarding the rate at which ribosomes move along the mrna ) . 40s and 60s are the ribosomal subunits , 80s is the monosome or complex of the two subunits , and m7 g is the cap , a modified nucleotide containing a methyl tag , present at the 5 end of the mrna . in cultured neurons , protein synthesis can be activated by several synaptic stimuli such as bdnf and dhpg . bdnf stimulates translation at synapses via the mtor and erk mapk pathways , and likely involves the modulation of translational initiation . moreover , bdnf activates the translation of arc and camkii mrnas ( yin et al . , 2002 ; schratt et al . , 2004 ; napoli et al . , 2008 ) , two fmrp targets ( zalfa et al . , 2003 ; park et al . , stimulation of neurons by bdnf or dhpg promotes dissociation of cyfip1 from eif4e at synapses , an event associated with initiation of protein synthesis . thus , fmrp - dependent translational repression in brain is mediated , at least in part , by cyfip1 ( napoli et al . , 2008 ) . activity dependent changes in fmrp phosphorylation is observed in dendrites and show a temporal correlation with the translational profile of select fmrp target transcripts ( narayanan et al . , 2007 ) . post - translational modifications of fmrp and or cyfip1 may account for such reversible control of local translation . finally , the temporal activity of fmrp seems also to be regulated by mglur signaling . mglur - ltd induces a transient , translation - dependent increase in fmrp that is rapidly reversed through degradation by the ubiquitin - proteasome pathway ( hou et al . , 2006 ) . nmda receptor - dependent synaptic stimulation promotes a redistribution of proteasomes from dendritic shafts to synaptic spines and thus provides a mechanism for local protein degradation ( bingol and schuman , 2006 ) . synaptic proteasomal activity seems to affect a large number of synaptic proteins including specific postsynaptic scaffolds whose turnover after ubiquitination is enhanced . interestingly , the long - term activity - dependent increases of camkii and psd-95 , encoded by two mrnas that are translationally repressed by fmrp ( zalfa et al . , 2003 ; hou et al . , 2006 ; muddashetty et al . , 2007 ; these findings suggest an interplay between mrna localization and fmrp degradation that is integrated with neuronal activity and directly regulated by the ubiquitin - proteasome system . fragile x mental retardation protein key target mrnas encode proteins involved in synaptic structure and function ( bagni and greenough , 2005 ; bassell and warren , 2008 ) . among them an interesting target candidate is arc / arg 3.1 ( zalfa et al . , 2003 ; park et al . , 2008 arc / arg 3.1 mrna translation is rapidly up - regulated following mglur stimulation , an effect that is not detected in the absence of fmrp . arc / arg 3.1 mrna synaptic translation has been proposed to be critical for synapse - specific ltd ( park et al . , 2008 ) . thus arc / arg 3.1 reduces the number of glur2/3 receptors leading to a decrease in ampar - mediated synaptic currents ( rial verde et al . , key ltd synaptic proteins whose upregulation would lead to the impaired hippocampal mouse ltd observed in fmr1 ko mice . ( 2004 ) proposed that a net loss of synaptic ampa and nmda receptors , possibly due to an increased internalization mediated by one or several ltd proteins , would contribute to a weakened spine and possibly the dysmorphogenesis observed in the fxs . fragile x mental retardation protein can specifically recognize its mrna targets in multiple ways : directly via rna structures such as g - quartets and/or g - rich and/or u - rich sequences , via bc1 rna , or micrornas ( bagni and greenough , 2005 ; bassell and warren , 2008 ) . it is unclear whether the alternative binding motifs or non - coding rnas are specific for different messengers or , rather , function as alternative modes to target the same mrnas during development or synaptic response . further work is needed to understand the functional consequences of impaired local protein synthesis in the developing brain and how this correlates with the autistic and fxs phenotypes . the cpeb is an rna binding protein that recognizes target rnas via specific sequences in their 3-untranslated regions . cpeb promotes dendritic delivery of mrnas along microtubules ( huang et al . , 2003 ) and polyadenylation - induced translation of mrnas in response to synaptic activity ( wu et al . , 1998 du and richter , 2005 ) ( figure 2 depicts key features of cpeb - controlled translation ) . in neurons , cpeb localizes to the cell body and postsynaptic sites and , within dendrites , is packaged in rnp granules that localize near synapses . in neurons cpeb targets a number of mrnas that encode proteins critical to synaptic plasticity ( du and richter , 2005 ; pique et al . , 2008 ) . disruption of cpeb stunts the development of dendritic arbors and prevents neurons from integrating into the functional visual system of xenopus laevis ( bestman and cline , 2008 ) . moreover , loss of cpeb activity in mice prevents the formation of some forms of synaptic plasticity and interferes with memory formation ( alarcon et al . ; mcevoy et al . , 2007 ; miniaci et al . , 2008 ) . cpeb knockout mouse exhibit reduced theta burst - induced ltp at schaffer collateral to ca1 pyramidal cell synapses of the hippocampus ( alarcon et al . , 2004a ) . in addition , these mice exhibit a deficit in extinction of memory , a phenomenon whereby behavioral responses diminish and eventually become extinct when there is no reinforcement of the memory ( berger - sweeney et al . although extinction requires the formation of new memories , the underlying mechanisms by which it occurs are probably distinct from those of memory acquisition and consolidation ( abel and lattal , 2001 ) . these data suggest an important role for cpeb in linking local protein synthesis and morphological plasticity to synaptic plasticity . some mrnas contain a cytoplasmic polyadenylation element ( cpe ) , which is bound by cpeb . cpeb also interacts with maskin ( or neuroguidin , a functionally related protein ) , which in turn interacts with the cap ( m7 g ) binding protein eif4e . such mrnas are translationally inactive because maskin inhibits the association of eif4e and eif4 g , another initiation factor that helps recruit the 40s ribosomal subunit to the 5 end of the mrna . cues such as nmda receptor activation stimulate the kinase aurora a , which phosphorylates cpeb , an event that causes poly(a ) tail elongation . poly(a ) binding protein ( pabp ) binds the newly elongated poly(a ) tail and recruits eif4 g . the pabp - eif4 g dimer helps to displace maskin from eif4e , allowing eif4 g to bind eif4e and initiate translation . identified growth hormone ( gh ) as one protein whose level is reduced 10-fold in the cpeb ko hippocampus ( zearfoss et al . , 2008 ) . gh mrna contains no binding sites for cpeb , and both gh mrna and pre - mrna are reduced in the ko versus wt hippocampus , suggesting that an mrna encoding a transcription factor that regulates gh gene expression is under the control of cpeb . indeed , cpeb controls the synthesis of c - jun , which regulates the expression of the gh gene . gh also induces ltp in hippocampal slices ; gh - induced ltp , like electrical stimulation - elicited ltp , is reduced at ca1 synapses of the cpeb ko mouse . moreover , ltp induced by gh or theta burst stimulation is reduced if polyadenylation is inhibited by the drug cordycepin ( 3-deoxyadenosine ) , which terminates adenylate polymers . these and other results suggest that gh acts in both autocrine and paracrine fashion to regulate plasticity through cpeb - mediated control of c - jun translation . the cpeb is also present in invertebrates , where it may act as a polyglutamine - containing prion - like factor to control plasticity ; in aplysia sensory neurons where cpeb rna expression has been disrupted by an antisense oligonucleotide , long - term facilitation is not maintained ( si et al . , 2003b ) . the isoform of aplysia neuronal cpeb differs from the vertebrate cpeb noted above in that it contains a high concentration of glutamine residues . polyglutamine can be found in proteins that resemble a prion , an infectious agent consisting of self - reproducing protein . the authors ( si et al . , 2003a , b ) suggested that this cpeb isoform could assume a prion - like structure following synaptic experience and form an indelible synaptic tag . if true , then cpeb , rather than the proteins derived from cpeb - regulated translation , would comprise a tag that distinguishes experienced from naive synapses . si et al . ( 2003a ) demonstrated that aplysia cpeb resembled a prion in that it was protease resistant and had a fast sedimentation rate in sucrose gradients . more compelling evidence came from experiments in transduced yeast , where aplysia cpeb assumed two forms : one that aggregated ( i.e. , prion - like ) and one that did not . very unexpectedly , the aggregated form of cpeb bound to rna and converted the nonaggregated form into an aggregated form . such epigenetic inheritance is a hallmark of prion formation and lead the authors to opin that synaptic activity could cause neuronal aplysia cpeb to become prion - like and stimulate the translation of rnas , cause it to alter its substrate specificity , or release some mrnas from a translationally dormant state . ( 2003a ) also suggested that once it becomes prion - like , cpeb would need no further stimulation to maintain activity . vertebrates contain three additional cpeb - like genes , all of which are expressed in the brain ( theis et al . , 2003 ) . two of these cpeb - like proteins contain some polyglutamine but they do not have a strong affinity for the cpeb binding site and do not regulate polyadenylation ( huang et al . , 2006 ) . although the relationship between vertebrate cpeb proteins and prions , if any , remains to be determined , a drosophila cpeb isoform , orb2 , contains polyglutamine and is important for long term memory ; when the polyglutamine is deleted , long term memory is disrupted ( keleman et al . , 2007 ) . while this study did not examine prion formation , or even mrna translation , it does point to the glutamine stretch of the cpeb isoform as being potentially important for memory formation several studies provide strong evidence linking mrna trafficking and dendritic protein synthesis to synaptic plasticity . relatively little is known about how these processes are interrelated , including coupling of synaptic signalling to the translational machinery , selective translation of specific mrna at synapses , and specific delivery of newly transcribed mrnas to activated synaptic sites . two molecules that connect receptor signalling to local protein synthesis in spines are cpeb and fmrp . cpeb localizes to postsynaptic sites where it regulates translation of proteins critical to synaptic plasticity ( du and richter , 2005 ; pique et al . , 2008 ) , remodeling of dendritic arborization and organization of the functional visual system ( bestman and cline , 2008 ) . cpeb ko mice exhibit deficits in synaptic plasticity and memory formation ( alarcon et al . , 2004b ; berger - sweeney et al . , 2006 ; mcevoy et al . , 2007 ; miniaci et al . , 2008 ) . these findings suggest an important role for cpeb in linking local protein synthesis and dendritic remodeling to synaptic plasticity . similarly , fmrp localizes to synapses where it regulates translation of proteins critical to synaptic plasticity and structural remodeling . fmr1 ko mice exhibit abnormal dendritic spines , cognitive deficits , aberrant synaptic plasticity in different areas of the brain , and a reduced number of mglur and ampa receptors . pharmacological or genetic manipulations that attenuate activity of group i mglurs ( dolen et al . , 2007 ) or exposure to an enriched environment increases ampar subunit abundance in the visual cortex ( restivo et al . , 2005 ; meredith et al . , 2007 ) and rescues , at least in part , the hallmark behavioral and synaptic abnormalities of the fragile x phenotype . dissection of the molecular mechanisms underlying these rescue experiments represent a starting point in our understanding of fmrp function and of the linkage between neuronal activity , receptor activation , and local protein synthesis . 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 . . main cause of acs is intra abdominal hypertension ( iah ) due to various factors such as abdominal surgery , ileus , intestinal obstruction , intraabdominal malignancy , and organomegaly . the most important clinical presentations of acs are symptoms due to abdominal distension , acute oliguria , and respiratory problems 1 . we report a patient with external compression of distal duodenal segment due to the metastatic lymph nodes and acute kidney injury ( aki ) as a result of acs . it was diagnosed with computed tomography ( ct ) and after surgical intervention his clinical status improved significantly . we report this case because to date , a case of acs due to metastatic prostate cancer has not been reported . a 78-year - old man admitted to emergency department with abdominal pain , distension , nausea , vomiting , and dyspnea for nearly 1 week and oliguria for 2 days . bilateral renal parenchymal echo increased consistent with the acute kidney failure and the bladder was empty . also there was no electrolyte disturbances that could contribute to kidney injury such as hypercalcemia , hypokalemia , hyperphosphatemia , or hyperuricemia . he had a history of prostate adenocarcinoma diagnosed 6 months ago but he did not accept the antineoplastic therapy performed for him . on physical examination , blood pressure was 115/80 mmhg , oral mucosa was wet and there was severe abdominal distension and tenderness . at admission laboratory values were as follows ; urea : 116 mg / dl , creatinine : 4.7 mg / dl , c - reactive protein ( crp ) : 77 mg / dl , chloride : 90 mmol / l , lactate dehydrogenase ( ldh ) : 556 /l , ph : 7.49 , pco2 : 35.9 mmhg , hco3 : 28 mmol / l . on urinalysis ; urine ph was < 5 and density was 1017 . on ct there were multiple lymph nodes in the abdominal cavity , also gastric and duodenal distension due to lymph node compression on distal duodenal segment ( fig.1 ) . intravesical pressure of the patient was 36 mmhg ( normal levels of intra - abdominal pressure : 05 mmhg ) . after nasogastric ( ng ) tube insertion , 8 l of gastric fluid discharged suddenly . after medical decompression intervention , urine output increased gradually and creatinine levels started to regress . but after the removal of ng tube , clinical status deteriorated again . therefore , surgical decompression ( gastrojejunostomy ) was performed . after decompression operation beside the clinical and radiological status ( fig.2 ) , laboratory values improved , but intravesical pressure was not measured again . after operation laboratory values were as follows ; creatinine : 2.4 mg / dl , crp : 27 mg / dl , ldh : 399 /l . during follow- up organ dysfunction due to iah is called as acs and it is a mortal condition especially for critically ill patients . acs is not a disease , however , it can have many causes and can develop during many disease processes . one of the most affected abdominal organs due to acs is kidney and aki is not rare in this condition . our case had an uncontrolled prostate cancer history and presented with abdominal pain , dyspnea and oliguria . at admission we determined oliguric aki and on ct gastric and duodenal distension due to metastatic lymph nodes one of the most important factors that contribute organ failure caused by iah is probably increased levels of proinflammatory cytokines . in a human model of iah induced by laparoscopic surgery , markers of both systemic inflammation and aki were increased . thus , it is not impossible to suggest that acs caused by iah is at least partly due to increased inflammation 2 . also in our patient during the acs period crp level was high and decreased with successful treatment . intestinal obstruction is a predictor of iah just as the fact that iah is a predictor of mortality . in the majority of ileus and/or intestinal occlusion cases there is iah and this is an early and important messenger of mortality in these conditions . intra- abdominal pressure ( iap ) higher than 2025 mmhg may cause acs and in this way acute functional loss of various abdominal and extra - abdominal organs . inflammatory and hemodynamic factors caused by iah may affect pelvic , thoracic , cranial and muscular areas beside abdominal organs 3,4 . 5 reported a patient with acs presented with aki caused by air - trapping and excessive auto - positive end - expiratory pressure during intubation and recovered after extubation . after abdominal surgeries iah is not rare and measurement of iap is thought to be a predictor of aki development for patients followed in intensive care units 6 . 7 reported a 72-year - old woman with acs due to gastric dilatation after the surgery of retroperitoneal sarcoma and presented with cardiorespiratory collapse and anuria . reported a pediatric patient from japan with acs due to hepatomegaly and neuroblastoma and after decompression operation and continuous hemodiafiltration his health status ameliorated 9 . iap may be detected by measuring the pressure of urinary bladder ( intravesical method ) and it well correlates with iap . treatment options of world society of the abdominal compartment syndrome are : improvement of abdominal wall compliance , evacuation of intra - luminal contents , evacuation of intra - abdominal space occupying lesions , optimization of fluid management and optimization of systemic and regional perfusion 3,4 . in the present case we detected aki due to acs in a patient with duodenal compression because of multiple metastatic intra - abdominal lymph nodes . during the follow - up period , especially for patients with malignant disorders with acute oliguria and respiratory problems who have abdominal distension , acs should be in mind as causative factor . for patients with acute oliguria and respiratory problems who have abdominal distension , acs and/or iah should be in mind as differential diagnosis among other possible disorders . the author declares that there is no conflict of interests regarding the publication of this article .

localization of mrnas to dendrites and local protein synthesis afford spatial and temporal regulation of gene expression and endow synapses with the capacity to autonomously alter their structure and function . emerging evidence indicates that rna binding proteins , ribosomes , translation factors and mrnas encoding proteins critical to synaptic structure and function localize to neuronal processes . rnas are transported into dendrites in a translationally quiescent state where they are activated by synaptic stimuli . two rna binding proteins that regulate dendritic rna delivery and translational repression are cytoplasmic polyadenylation element binding protein and fragile x mental retardation protein ( fmrp ) . the fragile x syndrome ( fxs ) is the most common known genetic cause of autism and is characterized by the loss of fmrp . hallmark features of the fxs include dysregulation of spine morphogenesis and exaggerated metabotropic glutamate receptor - dependent long term depression , a cellular substrate of learning and memory . current research focuses on mechanisms whereby mrnas are transported in a translationally repressed state from soma to distal process and are activated at synaptic sites in response to synaptic signals . key clinical messageorgan dysfunctions caused by intraabdominal hypertension is named as abdominal compartment syndrome ( acs ) . a patient with prostate cancer admitted with dyspnea and oliguria . after decompression his health status improved . for patients with malignant disorders presented with oliguria and respiratory problems who have abdominal distension , acs should be in mind .

lyme disease is a worldwide - distributed multisystem animal - borne disease , caused by borrelia burgdorferi ( bb ) sensu lato ( gram - negative spirochaetes ) which is a group of at least 12 closely related species . recent evidence shows that human cases of lyme disease may also be caused by more than one species of b. burgdorferi sensu lato , with rodents and small mammals as its animal reservoir . traditionally lyme disease is divided into four stages , and clinical manifestations may be both cutaneous and systemic and can have cardiovascular , neurological , and musculoskeletal involvement [ 46 ] . when lyme disease is treated with appropriate antibiotic therapy in the early stages , long - term outcomes are good . lyme disease is the most common vector - borne disease in the usa and europe with more than 300,000 new cases in the usa and 100.000 cases annually in europe ( estimated from available national data ) . however , this number is largely underestimated as case reporting is highly inconsistent and many infections go undiagnosed [ 1013 ] . global climate change expanding the range of tick vectors and an increase in the incidence suggest that this disease will remain an important health issue in the forthcoming decades . chronic lyme disease ( cld ) is considered a constellation of persistent symptoms in patients with or without evidence of bb infection [ 15 , 16 ] . some authors use the term cld only for patients who had been clinically diagnosed with lyme disease and have persisting symptoms lasting more than 6 months following antibiotic treatment . for this reason the term posttreatment lyme disease [ 17 , 18 ] or post - lyme disease syndrome [ 10 , 18 ] is often used . the category of probable lyme disease was recently added to the cdc surveillance case definition to describe patients with serologic evidence of exposure and physician - diagnosed disease in the absence of objective signs . recently , the term chronic arthropod - borne neuropathy ( can ) has been proposed as an alternative to chronic lyme disease , as calling the chronic illness there is growing and well - documented evidence to the concept of persistent bb infection in both animals [ 2131 ] and humans [ 3241 ] . recent evidence shows bb is able to escape from destruction by the host immune reactions , persist in host tissues , and sustain chronic infection and inflammation , despite aggressive antibiotic challenge [ 32 , 35 , 36 , 4244 ] . an estimated 20% of patients display recurrent symptoms after antibiotic treatment . a recent study showed that , at six months following antibiotic treatment , 36% of patients reported new - onset fatigue , 20% widespread pain , and 45% neurocognitive difficulties without evidence of depressive symptomatology . some studies have provided evidence on pleocytosis and the production of bb antibodies in the cerebrospinal fluid of patients with neuroborreliosis , while this is contradicted by other studies showing greater variability in such patients . when looking at the clinical and basic science research , it is apparent that persistent bb infection and associated inflammation and molecular mimicry mechanisms are associated with gradually increasing encephalopathy and gradually increasing mental symptoms . despite available evidence , both treatment - refractory infection and persistent symptoms that are related to bb infection remain subject to controversy in which national medical societies , patient advocacy groups , insurance companies , lawyers , doctors , the private health medical sector , and scientific journals all have become embroiled [ 10 , 5053 ] . the controversy is exacerbated by the lack of a clinical or microbiologic definition and the frequent occurrence of chronic symptoms in the general population . the lyme disease controversy can also be largely linked to the misconception that neurobehavioral effects of illness constitute evidence of nervous system infection . appropriate differentiation between neuroborreliosis ( nervous system borrelia burgdorferi infection ) and lyme encephalopathy ( altered nervous system function in individuals with systemic but not nervous system infection)or encephalopathies of other etiologies would lessen the controversy considerably , as the attribution of nonspecific symptoms to supposed ongoing central nervous system infection is a major factor perpetuating the debate . because of the absence of solid evidence on prevalence , causes , diagnostic criteria and treatment options , the role of autoimmunity to residual or persisting antigens , and the role of a toxin or other bacterial - associated products that are responsible for the symptoms and signs , cld remains a relatively poorly understood chronic disease construct . the role and performance of family medicine in the detection , integrative treatment , and follow - up of cld are not well studied either . the purpose of this paper is to describe the complexity of cld as a multidimensional chronic disease construct and its relevance to family medicine by means of a systematic literature review . this paper is a second paper in a series on chronic diseases that focus on complex chronic conditions . a systematic review method was used to document the complexity and multidimensionality of cld . in addressing the objective of this review , we used a parallel search strategy via medline , the cochrane library of systematic reviews , cinahl ( cumulative index to nursing and allied health literature ) , and pre - cinahl , complemented with a reference review of key articles . articles were selected if they dealt with ( 1 ) cld care complexity ; ( 2 ) cld case ( patient ) complexity ; ( 3 ) the complexity of cld quality assessment ; and ( 4 ) complexity of cld at the health system level . in addition the studies had to produce insights into the complexity of cld with relevance to family medicine . the search strategy included a combination of medical subject headings ( mesh ) terms with regard to lyme disease . we also scrutinised extra sources for further identification of studies by hand - searching the reference lists of all articles . by focusing on cld care , case , quality assessment , and health systems complexity , we further build on four major and interrelated components of complexity in chronic care that have been described by borgermans et al . . each of these components represents a range of elements that contribute to the picture of complexity in chronic care . two authors carried out independent screening of titles and abstracts using the specific inclusion and exclusion criterion reported on previously . we ordered the full text of all citations that met the eligibility criteria or appeared relevant or where relevance / eligibility was not clear from the abstract . in the final screening , two authors independently scrutinised the full texts of studies and recorded the reasons when articles were excluded resolving disagreements by discussion and when necessary referred to a third author . a tool - based assessment of the methodological quality of the studies was not part of this review . the flow diagram ( figure 1 ) shows our study selection process and the number of studies included . 72 studies provided insights on cld care complexity , 9 studies provided insights on cld case complexity , 2 studies provided insights on cld quality complexity , and 6 studies provided insights on cld health system complexity with relevance to family medicine . it is current practice for physicians to base themselves primarily on clinical signs and symptoms when cld is suspected . patients often can not recall being bitten by a tick or if erythema migrans has occurred . erythema migrans is pathognomonic and does not require any further laboratory investigations , but a careful analysis is recommended as a variety of unconventional histopathologic patterns may occur . in the case of no erythema migrans clinical manifestations are complemented by laboratory tests , including enzyme - linked immunosorbent assay ( elisa ) and western blot testing . although this two - tier testing is standard practice in both the united states and europe , the test kits generally differ . there is consistent evidence that the two - tier testing lacks sensitivity , can not distinguish between current and past infection , can not be used as a marker for treatment , is often dependent on subjectively scored immunoblots , and is considered expensive [ 64 , 65 ] . moreover , the diagnosis of cld based on clinical manifestations , serological findings , and detection of infectious agents often contradict each other [ 56 , 66 , 67 ] . for this reason a growing number of studies support two - tier testing followed by a sensitive and reliable dna sequencing for confirmation to support the diagnosis of cld [ 68 , 69 ] . other authors suggest the use of a single tier hybrid antigen immuno - pcr and detection of igg antibodies only . for cases with a high clinical suspicion of disease , the c6 peptide enzyme immunoassay ( eia ) is put forward as a stand - alone test or in the second tier of a 2-tiered algorithm . this assay is based on a peptide ( c6 ) , whose amino acid sequence reproduces a conserved , immunodominant region of a single protein of borrelia burgdorferi . overall , the use of pcr - based methods appears to be of importance because of the high sensitivity and specificity of these assays [ 7173 ] . however , pcr positivity in the absence of culture positivity following antibiotic treatment should be interpreted with caution since b. burgdorferi dna and mrna can be detected in samples long after spirochetes are no longer viable as assessed by classic microbiological parameters . another novel method including culturing spirochetes from the serum of patients uses a two - step preenrichment process , followed by immunostaining with or without polymerase chain reaction ( pcr ) analysis . in patients who complied with the strict cdc surveillance case definition for lyme disease the procedure resulted in positive cultures in 47% at 6 days and 94% at week 16 . the centers for disease control and prevention has however raised concerns about false - positive results caused by laboratory contamination and the potential for misdiagnosis . some authors put forward the measurement of activity of two lysosomal exoglycosidases , -fucosidase ( fuc ) and -galactosidase ( gal ) , in the serum as potential markers of ld . recent evidence has shown that the presence or absence of chronic lyme borreliosis may be objectively adjudicated by tissue examinations which demonstrate or which fail to show pathogenic microbes in patients who have received a full course of antibiotics . the use of spect scans of the brain using tc and standard nuclear imaging techniques is considered an objective measure of abnormalities present in patients with otherwise difficult to objectify clinical findings . brain spect scans are abnormal in most patients with chronic lyme disease , and these scans can be used to provide objective evidence in support of the clinical diagnosis . overall , studies highlight the need for standardization in diagnostic ( serological ) testing , as well as the need for studies that discriminate between active disease and past infection . a factor that complicates the diagnosis of cld is that it does not present with isolated subjective symptoms . in undiagnosed patients or posttreatment , bb may mimic the symptoms and signs of other diseases disorders [ 80 , 81 ] and for this reason is called the illness with a thousand faces . these symptoms may include fatigue , insomnia , widespread pain , arthritis , chronic eye lid edema , cataract , dermatitis , cognitive complaints , bilateral hearing loss , adult respiratory distress syndrome , palpitations , tachycardia , syncope , tremors , epileptic crises , depression , anxiety , panic attacks , catatonia , and psychosis , amongst others [ 93 , 94 ] . lyme disease might become misdiagnosed as fibromyalgia , chronic fatigue syndrome [ 96 , 97 ] , amyotrophic lateral sclerosis , autism spectrum disorder , parkinson 's disease , rheumatoid arthritis , multiple chemical sensitivity , guillain - barre syndrome ( gbs ) , and dementia , as well as other numerous neurological [ 101 , 102 ] and psychiatric disorders [ 88 , 103 ] . overall , studies show the need for a careful differential diagnosis in patients with suspected cld and persistent complaints . the diagnosis of cld is even more complex when tick - borne coinfections occur in association with lyme disease , which is the rule rather than the exception . routine laboratory tests exhibit varying reported sensitivity and are usually unhelpful in diagnosis , as serology fails in terms of specificity and/or sensitivity . clinically relevant coinfections include bartonella species , babesia , anaplasma , rickettsia , yersinia enterocolitica , chlamydophila pneumoniae , chlamydia trachomatis , mycoplasma pneumoniae , and tick - borne encephalitis virus , amongst others [ 108 , 111 ] . a particular condition that gets growing attention in patients with cld is morgellons disease . morgellons disease is an emerging skin disease characterized by formation of dermal filaments associated with multisystemic symptoms . recent studies show that bb spirochetes were detected in the dermatological specimens from study patients , providing evidence that morgellons disease is associated with an infectious process [ 112115 ] . overall , studies highlight the importance of coinfections since they can complicate the diagnostic process and their pathological synergism can exacerbate cld or induce similar disease manifestations . treatment options are complicated since the population of individuals reporting cld are heterogeneous with guidelines contradicting each other . recommendations about the type and duration of treatments in patients with cld have no factual basis , although prolonged courses of antibiotics are likely to be helpful . recent biostatistical review reveals that retreatment has meaningful clinical benefit in patients who had prior antibiotic treatment [ 116 , 117 ] , with the use of longer ( parenteral ) antibiotic therapy often to be justified . doxycycline is often the preferred agent for oral treatment because of its activity against other tick - borne illnesses , but recent evidence shows that doxycycline - treatment only does not always lead to clinical improvements in either the persistent symptoms or quality of life in patients with cld . this finding can potentially be explained since single antibiotic use often fails to address the different morphological forms of bb as well as biofilm formations in patients with cld . studies indicate the need to include cell wall and cystic and intracellular drugs in any treatment as the different morphological forms of bb display differences in sensitivity to antibiotic treatment . as coinfections are present in most patients , the use of specific antibiotics is required . tetracyclines and macrolides are often used , with quinolones for alternative treatment , particularly gemifloxacin . for bartonella henselae , chlamydia trachomatis , and chlamydophila pneumoniae the combination with rifampicin is recommended . recent evidence shows that novel therapeutic targets for the treatment of the disease should acknowledge a central role of the neutrophil - activating protein a ( napa ) of bb in promoting both regulatory t - cell response and immune suppression in the cerebrospinal fluid of patients with chronic lyme borreliosis . overall , studies show that various therapeutic regimens are used in patients with cld reflecting the need for individualized approaches . the vector model of complexity ( vmc ) is a useful model to describe case complexity in patients with cld . the vector model proposes that the complexity of an individual patient arises out of interactions between different domains : biology , genetics , socioeconomics , environment , behaviour , culture , and the health system . in a chronic condition such as cld , complexity in cld along the biology / genetics axis is important since gender distribution in patients with lyme borreliosis has recently been demonstrated . patients with cutaneous manifestations of lyme borreliosis tend to be predominantly female , whereas those with noncutaneous manifestations are predominantly male . complexity in cld is introduced along the environment axis with a growing number of studies to document the important relationship between the increase in outdoor activities in wooded areas and the incidence of lyme disease . special environmental risk factors include the presence of deer ticks in the home environment , ground cover containing moist humus , and leaf litter in the yard . distribution areas as well as host and vector ranges of lyme borreliosis agents turned out to be much wider than previously thought [ 2 , 124 ] . another element from the environment that contributes to the complexity of cld is that the variety of bb genospecies leads to distinction in clinical manifestations of lyme borreliosis ( lb ) [ 125 , 126 ] . at present , the risk of exposure to multiple pathogens from a single tick bite and the sources of coinfected ticks are not well understood . complexity in cld is also introduced along the behavioral axis as cld has considerable implications on daily life . patients report a significant and severe decline in health status associated with chronic lyme disease . studies have shown that a history of severe , long - term , premorbid , and psychological stress is associated with increased incidence of chronic physical symptoms in lyme disease patients . traumatic psychological experiences predating onset of lyme disease symptoms may play an important etiologic role in the chronicity of these symptoms . as a consequence , the burden of cld is likely to be underestimated as a consequence of the inadequate recognition of the connection between mental and physical health . the complexity of quality assessment is reflected by the lack of tools at the present time that can assess the quality of care delivered to patients with cld . a limited number of quality indicators at the structure , process , or outcome level of care for patients with cld exist , which is in part a consequence of the lack of research on ( the construct of ) cld . in addition , publications that describe approaches to patient involvement in quality indicator development are scarce and to our knowledge nonexisting for patients with cld . there is an abundance of studies on health seeking behaviour highlighting the importance of health system characteristics and their influence on an individual 's behaviour at a given time and place [ 129 , 130 ] . these influences include the financing of care , access to care , coordination mechanisms , and existing stigma on unexplained medical conditions and values and norms . a large survey conducted in the usa in patients with cld demonstrated extensive delays in obtaining an initial diagnosis and having poor access to healthcare [ 46 , 131 ] . negative experiences are associated with reports of dismissive , patronizing , and condescending attitudes in health care providers . studies show that consultations with complementary and alternative medicine ( cam ) practitioners and use of cam therapies are common , representing a cost to patients to be exorbitant and prohibitive . we have outlined the importance of case , care , quality assessment , and health system complexity in patients with cld . the majority of studies focus on cld care and case complexity with a minority of studies to report on cld quality assessment and health system complexity . while specialists are an essential element of the total health care continuum , the majority of patients with cld will continue to access the health care system through family physicians , with hospitalization due to lyme disease to be rare . a range of persistent misconceptions in family medicine exists , ranging from the reliability of available diagnostic tools , the signs and symptoms of nervous system involvement , the importance of coinfections , the appropriate choice and duration of antimicrobial therapy , the importance of jarisch - herxheimer reaction after the commencement of treatment with antibiotics , the curability of the infection , and the cause of symptoms that may persist in some patients after treatment [ 136138 ] . lyme literate family physicians seem to be rare which provides argument to the diagnosis and treatment of cld within the context of a broad - based , multidisciplinary approach to determining the best approach . comprehensiveness is the ability of the family physician to address a broad range of patient problems , whether or not the conditions are within the traditional domain of the specialty in which the physician is trained . referral to lyme literate specialists is essential both to meet the patient 's needs and to establish an integrated care network where responsibilities and tasks are shared . an effective response to the health needs of those with cld will especially require family physicians and specialists to expand their collaborative efforts and knowledge of each other 's practices and treatments . collaboration between family physicians , dieticians , and physiotherapists is considered important since healthy lifestyles are essential in the recovery of patients with cld . another component of the care responsibility of family physicians is ensuring that patients receive preventive interventions . family physicians can contribute to public health interventions in high - incidence age groups focusing on accurately communicating risk , improving knowledge both of lyme disease symptoms and of ticks that carry the disease , as well as teaching preventive behaviors to help reducing tick - borne illness rates . research has shown that patients ' knowledge of tick - borne illnesses is poor and the frequency of practicing preventive behaviors is low . it must be noted , however , that a growing number of patients consult medical sources on the internet with the aim of increasing their health literacy on the subject . family physicians must encourage and support their patients in search of reliable sources of information . the controversy on cld needs to be solved because of the heavy burden of illness associated with cld in patients with or without evidence of bb infection . there is a need for the development and establishment of new clinical diagnostic tools with increased accuracy , sensitivity , and specificity , as well as novel treatment approaches that may reduce the burden of illness and concomitant costs posed by cld . family physicians have a crucial role to play in the prevention and treatment of the disease fostering an integrative multidisciplinary approach to care . poly(3-hydroxybutyrate ) , p(3hb ) is a biodegradable thermoplastic polyester accumulated intracellularly by many microorganisms under unfavorable growth conditions . the high production cost of p(3hb ) can be decreased by strain development , improving fermentation and separation processes [ 24 ] , and/or using a cheap carbon source . in p(3hb ) production , about 40% of the total production cost is contributed by the raw material , whereby the cost of carbon feedstock alone accounts for 70 to 80% of the total raw material cost [ 6 , 7 ] . therefore , the utilization of renewable and sustainable substrates for the production of p(3hb ) has become an important objective for the commercialization of bioplastics . a lot of research have been carried out to discuss and propose the utilization of renewable biomass to replace commercial sugars as carbon source in order to reduce the production cost of p(3hb ) [ 812 ] . recently , we reported on the use of oil palm frond ( opf ) juice as the novel and renewable feedstock for the production of p(3hb ) . we demonstrated that opf juice is a good substrate for the production of p(3hb ) from wild - type cupriavidus necator ( ccug52238 ) , with better yield of product formation in comparison to technical grade sugars . this can be explained by the presence of minerals and nutrients in the opf juice which are essential for bacterial growth during fermentation . apart from contributing to higher product formation and microbial growth , the use of opf juice is advantageous compared to the other lignocellulose - based sugars due to the ease in its processing wherein no harsh pretreatment steps and enzymatic treatment will be needed in order to obtain the sugars . in our report , c. necator is well known as polyhydroxyalkanoate ( pha ) producer and its ability to accumulate pha more than 50 wt.% has been previously reported [ 8 , 14 ] . in general , p(3hb ) accumulation is favored by an excess carbon source and inadequate supply of macrocomponents such as nitrogen , phosphate , and dissolved oxygen or microcomponents such as magnesium , sulphate , iron , potassium , manganese , copper , sodium , cobalt , tin , and calcium [ 7 , 15 ] . moreover , it was also reported that the accumulation of p(3hb ) in microorganisms were influenced by several physical parameters including ph and agitation speed [ 1619 ] . in order to make the production of p(3hb ) feasible for industrial application , it is crucial to have high p(3hb ) production yield . in this study , we investigated the effect of initial medium ph , agitation speed , and ammonium sulfate ( nh4)2so4 concentration on p(3hb ) production from c. necator ( ccug52238 ) utilizing opf juice in shake flasks fermentation with the aim to clarify the effect of each fermentation parameter on the microbial growth and p(3hb ) formation . the effect of dissolved oxygen tension ( dot ) level on cell growth and p(3hb ) production was investigated by conducting batch fermentation in 2-l - bioreactor . p(3hb ) produced from this study was then characterized for its thermal and mechanical properties . in this study , c. necator ( ccug52238 ) was obtained from the culture collection , university of goteborg , sweden and used for the production of p(3hb ) . the inoculum preparation , media , and cultivation conditions for c. necator ( ccug52238 ) are similar to those reported by zahari et al . p(3hb ) biosynthesis was carried out through one - stage cultivation fermentation in shake flasks . opf juice in this study was obtained by pressing fresh opf following the method described earlier . opf juice which comprises fructose , glucose and sucrose was diluted from stock ( 55 g / l of total initial sugars and used as carbon sources throughout the study period . in order to study the effect of culture medium initial ph on biosynthesis of p(3hb ) , the initial ph value of each msm and opf juice was adjusted to ph 6.08.0 using 2 m naoh prior to autoclaving . another set of experiment was conducted to study the effect of agitation on p(3hb ) production by testing several agitation speed at 180 , 200 , 220 , 240 , and 260 rpm . for the effect of ammonium sulfate concentration , various concentrations of ( nh4)2so4 in the range of 02.0 the cultures were incubated at 30c under aerobic condition , and all experiments were conducted in duplicates . in order to study the effect of dissolved oxygen tension ( dot ) on cell growth and p(3hb ) production profile under the optimized condition obtained from the shake flask study , batch experiment was conducted in 2-l - bioreactor ( 1 l working volume ) at different dot levels of 20 , 30 , 40 , and 50% . 100 ml of pregrown cells from growing stage were transferred into 900 ml msm in 2l bioreactors ( sartorius , germany ) supplemented with opf juice at 30% ( v / v ) dilution . g / l of initial total sugars concentration was autoclaved separately prior to addition with the msm medium . the msm compositions were prepared as previously reported by zahari et al . , except that 0.5 the temperature inside the bioreactor was set at 30c , while dot level was set at various concentrations of saturation throughout the fermentation using cascade mode and supplied with air at 1.0 vvm . the ph value during fermentation was controlled at ph 7.0 0.05 by 2 m naoh / h2so4 . samples were withdrawn every 5 h for the period of 50 h for the determination of cdw , p(3hb ) concentration , residual sugars , and ammoniacal nitrogen ( nh3-n ) content . residual sugars concentration , cell dry weight measurement , and p(3hb ) analysis were done as previously described by zahari et al . . the samples from the bacterial fermentations were taken at the end of the cultivation period to measure the total dry weight and p(3hb ) content . each sample was centrifuged at 11,000 g for 5 min at 4c ( thermo fisher scientific , nc , usa ) and the solids were washed with distilled water and centrifuged for two consecutive times . dry weight measurements were carried out by drying the solids at 50c and cooling in a desiccator to constant weight . the p(3hb ) content and composition in the lyophilized cell were determined using the gas chromatography ( shimadzu gc-2014 ) . approximately , 20 mg of lyophilized cells were subjected to methanolysis in the presence of methanol and sulfuric acid [ 85% : 15% ( v / v ) ] . the organic layer containing the reaction products was separated , dried over na2so4 , and analyzed by gc according to the standard method using an id - bp1 capillary column , 30 m 0.25 mm 0.25 m film thickness ( sge ) . residual ammoniacal nitrogen ( nh3-n ) content analysis was done using nessler method according to standard procedures ( hach , usa ) which was previously described by zakaria . samples with appropriate dilution factor were filled to 25 ml in the sampling bottles . three drops of mineral stabilizer were added into solution and the bottle was inverted for several times . three drops of polyvinyl alcohol also were added into the solution and mixed well with inversion several times . lastly , 1.0 ml of nessler reagent was added to the mixtures and mix thoroughly by inversion . the standard solution was prepared by replacing the samples with deionised water as blank sample . the sample solution was determined at the wavelength ( ) 425 nm using dr/4000 spectrophotometer by following the manufacturer , instructions ( hach , usa ) . residual sugars were determined by a high performance liquid chromatography ( hplc ) ( agilent series 1200 , usa ) using the supelcosil lc - nh2 column ( sigma aldrich ) ( 25 cm 4.6 mm i d , 5 m particles ) with a ri detector operated at 30c . the mobile phase was acetonitrile : water ( 75% : 25% ) at a flow rate of 1.0 ml / min . the components were identified by comparing their retention times with those of authentic standards under analytical conditions and quantified by external standard method . thermal properties of the polymer were determined by differential scanning colorimetry ( dsc ) ( ta instruments ) . for dsc analysis , 57 mg of homopolymer samples were weighed and heated from 20 to 200c at heating rates 10c / min and held for 1 min . the second scan was used in reheating the samples at the same heating rates and was used in evaluating the thermal properties of the biopolymer . the tensile strength , young 's modulus and elongation to break were determined by using instron universal testing machine ( model 4301 ) at 5 mm / min of crosshead speed . mechanical tensile data were calculated from the stress - strain curves on average of five specimens . the effect of initial medium ph on biosynthesis of p(3hb ) from opf juice was studied by varying the ph between ph 6.0 and 8.0 due to the fact that c. necator can tolerate and produce pha at the aforementioned ph range . suitable initial medium ph is crucial for the cell growth and p(3hb ) accumulation by c. necator ( ccug52238 ) . as shown in table 1 , increasing the initial medium ph value at intervals of 0.5 units affected both the cell growth and p(3hb ) production . both the cell growth and p(3hb ) content were increased when the initial medium ph was increased from ph 6.0 to ph 7.0 , that is , from 6.42 g / l to 8.57 g / l for cdw and 20 wt.% to 34 wt.% for p(3hb ) content , respectively . however , further increase of initial medium ph above ph 7.0 decreased both the cdw and p(3hb ) content . from the results , it can be concluded that ph 7.0 was the optimum initial medium ph for the growth and biosynthesis of p(3hb ) by c. necator ( ccug52238 ) in which , 8.57 g / l of cdw and 34 wt.% of p(3hb ) accumulation was recorded . the optimal ph for the cell growth and p(3hb ) accumulation in this study was similar to those reported in the literature . it was reported that the optimum ph for growth and p(3hb ) production by a. eutrophus was ph 6.9 and that a ph of 5.4 inhibited its growth . on the other hand , lowest cdw and p(3hb ) content , 4.02 g / l and 10 wt.% , respectively , were obtained at ph 8.0 . lowest cell growth and p(3hb ) accumulation at this initial ph value were obtained might due to alkaline condition which could affect the p(3hb ) production . , recommended a ph range of between 6.0 to 7.5 for microbial growth and p(3hb ) production . although p(3hb ) production can be controlled by precisely manipulating the medium ph , it has been reported that ph values other than 7.0 affected p(3hb ) production . these results suggested that p(3hb ) production is sensitive to the ph of cultivation . table 2 displays the effect of agitation speed on biosynthesis of p(3hb ) using opf juice as substrate in shake flasks experiment . it is interesting to note that both cell growth and p(3hb ) production showed an increasing trend with the agitation speed up to 220 rpm . for the agitation speed of more than 220 rpm agitation not only provides mixing and homogeneous cell and heat dispersion in the fermentation broth , but also better aeration for the cells by increasing the oxygen transfer rate throughout the fermentation medium . generally , slower agitation speed may cause the possibilities of cells aggregation , making the culture medium more heterogeneous . this may cause the cell growth to be decreased and thus affecting the production of p(3hb ) . on the other hand , increasing agitation speed higher than its optimal level may reduce the p(3hb ) formation , and hence , the cdw . this is due to the fact that pha is only produced and stored as granules in the cell cytoplasm by microorganisms when they are under stress conditions , for example when there is limitation of nutrient or electron acceptor such as oxygen . in our study , the best condition for the biosynthesis of p(3hb ) is at moderate agitation speed which is at 220 rpm with the highest cdw and p(3hb ) content reaching up to 9.42 ( nh4)2so4 has been widely used as the inorganic nitrogen source for the biosynthesis of p(3hb ) by c. necator . it is important to optimize nitrogen content in fermentation medium as p(3hb ) accumulation in the microorganisms can be triggered when one of the nutrients ( n , p , mg , and o2 ) in the mineral salt is limited in the presence of excess carbon source [ 14 , 15 ] . the effect of different ( nh4)2so4 concentrations on biosynthesis of p(3hb ) by c. necator ( ccug52338 ) from opf juice is summarized in table 3 . in overall , it was observed that cdw was increased when ( nh4)2so4 concentration increased from 0 to 2.0 on the other hand , p(3hb ) accumulation decreased with the increase of ( nh4)2so4 concentration . highest p(3hb ) accumulation at 44 wt.% was achieved when there was no addition of ( nh4)2so4 , in the culture medium . however , unsatisfactory cell growth that is , 5.25 g / l of cdw was observed in the experiment . based on the results , it was also found that ( nh4)2so4 concentration at 0.5 g / l was the optimal concentration for p(3hb ) accumulation and cdw formation , giving 42 wt.% and 8.31 these results corroborate to the literature , which reported that p(3hb ) formation predominantly occurs under - nitrogen and oxygen - limited conditions [ 14 , 15 , 28 , 29 ] . it was discussed that excess nitrogen source may restrict acetyl - coa from entering p(3hb ) production pathways and otherwise channelling into tca cycle for biomass production [ 15 , 21 , 28 ] . biosynthesis of p(3hb ) was then carried out in shake flask under the optimized conditions : initial ph medium , 7.0 ; agitation speed , 220 rpm and ( nh4)2so4 concentration , 0.5 g / l . under these conditions , the p(3hb ) produced from this study is 40% higher compared to the p(3hb ) produced under nonoptimized condition as shown in our previous study . the results presented herewith demonstrated that ph , agitation speed , and nitrogen concentration indeed plays an important role in the p(3hb ) production by c. necator ( ccug52338 ) utilizing opf juice . biosynthesis of p(3hb ) from opf juice by c. necator ( ccug52338 ) was carried out through batch cultivation process in 2-l bioreactor . the effect of dot level in the bioreactor was studied for do concentrations of 20 to 50% and the results are shown in table 4 . it was observed that cdw was increased when dot level increased from 20 to 50% . on the other hand , p(3hb ) g / l ) and p(3hb ) content ( 46 wt.% ) were achieved at 50 and 20% dot level , respectively . based on the result , it was found that dissolved oxygen concentration in the fermentation medium improved the cell growth ; however , p(3hb ) accumulation was found to be increased towards oxygen limitation . this result suggested that appropriate level of oxygen is needed for cell development , and oxygen depletion was favorable for p(3hb ) accumulation . as shown in table 4 , p(3hb ) accumulation was tripled at lower dissolved oxygen concentration ( 20% ) compared to the higher ones ( 50% ) . this might be due to the fact that insufficient supply of oxygen to the bacteria may decrease oxidation of nadh and lead to p(3hb ) biosynthesis [ 15 , 21 , 28 ] . a similar observation was obtained in our previous study on the effect of different ( nh4)2so4 concentration on p(3hb ) production using opf juice in shake flask experiment . these results indicate that both nitrogen and oxygen limitation do not improve cell biomass development , but markedly improve the p(3hb ) accumulation . therefore , it can be suggested that besides nitrogen depletion , oxygen limitation is also important in getting the optimal level of p(3hb ) accumulation . in order to study cell biomass and p(3hb ) production profile by c. necator ( ccug52238 ) , batch cultivation process was carried out using opf juice in 2-l bioreactor with aeration supplied at 30% dot level and the results were depicted in figures 1(a ) and 1(b ) . it was observed that the culture entered the exponential phase after a lag of 15 h , and nitrogen was completely consumed within 35 h. highest cdw ( 11.37 g / l ) and p(3hb ) content ( 44 wt.% ) were achieved at 45 hr cultivation period . the biomass yield ( yx / s ) and p(3hb ) yield ( yp / s ) were 0.81 g biomass / g sugars consumed and 0.36 g p(3hb)/g sugars consumed , respectively . the maximum p(3hb ) productivity was 0.11 g / l / h . almost similar p(3hb ) content with some improvement in cell growth g / l ) and biomass yield ( 0.81 g biomass / g sugars consumed ) obtained in fermentor compared to shake flask were due to different conditions which prevail in the shake flasks and fermentor ; some of these conditions include aeration , agitation , and temperature . in fermentor , aeration was supplied via air sparging , and agitation is provided by an impeller or by the motion imparted to the broth ( liquid phase ) by rising gas bubbles . temperature is maintained at a constant and uniform value by circulation of cooling water through coils in the vessel or in a jacket surrounding the vessel . compared to our previous studies in shake flasks using technical grade sugars , batch studies in 2-l bioreactor using renewable sugars from opf juice showed superior results in c. necator ccug52238 probably due to the additional components in the opf juice that improve the fermentation performance . an almost similar observation was reported by koutinas et al . when wh and fe were used as renewable feedstock for p(3hb ) production . it was reported that the consumption of various carbon sources ( carbohydrates , amino acids , peptides ) presented in the feedstock resulted in high growth yields ( up to 1.07 g cells / g glucose ) as related to glucose . as shown in figures 1(a ) and 1(b ) , the microbial growth is mainly associated with ammoniacal nitrogen consumption . for the first 35 h , lower sugars consumption by c. necator was observed . the sugars consumption within the time range was only 8.03 g / l which is half of the total sugars in the culture broth . on the other hand , the nh3-n was found to be decreased drastically from initial and completely exhausted after 35 h of cultivation period . this result indicates that at initial , the microbial growth was mainly attributed by the consumption of nitrogen sources from ( nh4)2so4 supplied earlier as one of the medium composition in bioreactor . in addition to that , other organic compounds such as amino acids , carbohydrates , and other minerals which were previously characterized in the opf juice could be used as supplementary growth substrates by the bacterium . after that , the cell growth was mainly contributed by the cell expansion due to p(3hb ) accumulation inside the cells . it can be seen that the p(3hb ) accumulation was doubled that is , 20 wt.% to 40 wt.% , from 35 h to 40 h of cultivation period . from sugars consumption and p(3hb ) profiles , it can be observed that the detectable depletion of sugars in the medium from 35 h onwards can be associated with p(3hb ) accumulation . these results are in agreement with the findings of other researchers that reported p(3hb ) accumulation is favored by an excess of carbon source and limited supply of macrocomponents such as nitrogen and dissolved oxygen [ 7 , 16 , 29 ] . it is interesting to note that c. necator ccug52238 completely utilized the glucose in the opf juice . regardless of fructose ( due to too low concentration of fructose in the medium ) , it seems like the bacterium preferred to consume glucose compared to sucrose . glucose consumption rate by c. necator ccug52238 was much higher at 0.33 g / l / h , compared to that of sucrose that is , 0.049 g / l / h . the mechanical and thermal properties of the homopolymer produced in 2-l bioreactor are shown in table 5 . the mechanical and thermal properties of p(3hb ) obtained in this study showed an almost similar properties to those reported in the literature . for instance , the tensile strength and elongation to break for p(3hb ) produced in this study were 40 mpa and 8% , respectively , and it was comparable to the p(3hb ) produced from pure fructose . the melting temperature , tm of p(3hb ) obtained from opf juice ( tm = 162.2c ) , was slightly lower compared to the melting point 177c reported for p(3hb ) produced from pure fructose and other renewable sugars such as maple sap . this could be influenced by other properties of the p(3hb ) such as molecular weight . it has been reported that the molecular weight of p(3hb ) produced is mainly influenced by the type of bacterial strain , substrate , growth rate , and production temperature [ 15 , 29 , 32 ] . this study demonstrated that higher cell growth and p(3hb ) accumulation can be obtained by culturing cupriavidus necator strain ccug52238 at optimized condition using opf juice as the sole renewable carbon source . under the optimal conditions , the highest cell weight was 9.31 0.13 g / l with 45 1.5 wt.% of p(3hb ) contained in the cells , accounts of 40% increment for p(3hb ) content compared to the nonoptimized condition . g / l and p(3hb ) content of 44 wt.% . in the meanwhile , thermal and mechanical characterization of the p(3hb ) obtained from opf juice showed almost similar properties to those reported in the literature . it is worth to mention that this study may contribute to the process development for p(3hb ) production from renewable opf juice in pilot and industrial scale . furthermore , since opf is an abundant solid waste at oil palm plantation and is currently underutilized , it has a great potential to be used as sustainable , renewable , and cheap fermentation feedstock for the production of p(3hb ) .

lyme disease has become a global public health problem and a prototype of an emerging infection . both treatment - refractory infection and symptoms that are related to borrelia burgdorferi infection remain subject to controversy . because of the absence of solid evidence on prevalence , causes , diagnostic criteria , tools and treatment options , the role of autoimmunity to residual or persisting antigens , and the role of a toxin or other bacterial - associated products that are responsible for the symptoms and signs , chronic lyme disease ( cld ) remains a relatively poorly understood chronic disease construct . the role and performance of family medicine in the detection , integrative treatment , and follow - up of cld are not well studied either . the purpose of this paper is to describe insights into the complexity of cld as a multidimensional chronic disease construct and its relevance to family medicine by means of a systematic literature review . factors influencing poly(3-hydroxybutyrate ) p(3hb ) production by cupriavidus necator ccug52238 t utilizing oil palm frond ( opf ) juice were clarified in this study . effects of initial medium ph , agitation speed , and ammonium sulfate ( nh4)2so4 concentration on the production of p(3hb ) were investigated in shake flasks experiments using opf juice as the sole carbon source . the highest p(3hb ) content was recorded at ph 7.0 , agitation speed of 220 rpm , and ( nh4)2so4 concentration at 0.5 g / l . by culturing the wild - type strain of c. necator under the aforementioned conditions , the cell dry weight ( cdw ) and p(3hb ) content obtained were 9.31 0.13 g / l and 45 1.5 wt.% , respectively . this accounted for 40% increment of p(3hb ) content compared to the nonoptimized condition . in the meanwhile , the effect of dissolved oxygen tension ( dot ) on p(3hb ) production was investigated in a 2-l bioreactor . highest cdw ( 11.37 g / l ) and p(3hb ) content ( 44 wt.% ) were achieved when dot level was set at 30% . p(3hb ) produced from opf juice had a tensile strength of 40 mpa and elongation at break of 8% demonstrated that p(3hb ) produced from renewable and cheap carbon source is comparable to those produced from commercial substrate .

a substantial body of evidence indicates , that low - grade inflammation in the vascular endothelium promotes atherogenesis [ 1 , 2 ] . this is indirectly evidenced by the fact that several inflammatory markers are linked to an increased risk of atherosclerosis . high - sensitive crp ( hscrp ) has been reported to associate with an increased risk of myocardial infarction , stroke , sudden death from cardiac causes , and peripheral arterial disease . in addition , hscrp has been shown to predict the first cardiovascular event better than low - density lipoprotein cholesterol ( ldl - c ) . recruitment of inflammatory cells from the circulation and their transendothelial migration is one of the earliest phases of atherogenesis . vascular endothelium expresses cellular adhesion molecules ( cams ) in response to several inflammatory stimuli . the evidence for the role of cams in the pathogenesis of atherosclerosis has emerged from experimental studies which have demonstrated the focal expression of adhesion molecules in atherosclerotic plaques [ 46 ] . selectins and their ligands are involved in the first steps in leukocyte adhesion at sites of endothelial injury as they mediate the rolling and tethering of leukocytes in the vascular wall [ 1 , 2 ] . intercellular adhesion molecule-1 ( icam-1 ) and vascular cell adhesion molecule-1 ( vcam-1 ) induce firm adhesion of inflammatory cells to the vascular surface . overall , both soluble icam-1 ( sicam-1 ) and soluble vcam-1 ( svcam-1 ) have been linked to increase risk of atherosclerosis [ 79 ] . apoe is transported in lipoproteins , and it regulates the hepatic uptake of remnant lipoproteins and facilitates cholesterol efflux from foam cells . in addition , apoe has a central role in inflammation and in mediating the central nervous system response to injury [ 1113 ] . there are three common apoe isoforms , designated e2 , e3 , and e4 , which are encoded for by distinct alleles on human chromosome 19 . apoe isoforms are distributed in six different phenotypes e2/2 , e2/3 , e3/3 , e4/2 , e4/3 , and e4/4 . the presence of e4 isoform is associated with coronary heart disease and with the early onset of alzheimer 's disease . so far , there are sparse data on apoe polymorphism in relation to the parameters of inflammation . mnttri et al showed that dyslipidemic men with e4/4 , e4/3 , and e4/2 phenotypes exhibit lower crp values than men carrying other apoe phenotypes . in a recent report by mrz et al , carriers of apoe2 allele and apoe3/3 homozygotes had significantly higher crp than individuals with the genotypes apoe3/4 and apoe4/4 . in dyslipidemic men and women , c - reactive protein has been reported to be lower in subjects with one or two copies of e4 allele than in those with zero e4 allele . paradoxically , e4 allele has been associated with an enhanced inflammatory reaction after cardiopulmonary bypass . in line , transgenic mice expressing the e4 allele had greater systemic and brain elevations of proinflammatory cytokines than e3 carriers . we hypothesize that the apoe phenotype , as a critical component in inflammatory processes , may influence the levels of inflammation markers . hdl has been demonstrated to exhibit antiinflammatory properties by inhibiting cytokine induced expression of adhesion molecules [ 2123 ] . in contrast , acute inflammation process may decrease hdl concentration and thus compromise its antiatherogenic function [ 24 , 25 ] . therefore we studied the effects of apoe phenotype on the levels of crp and scams on a sample of finnish low - hdl families and in normolipidemic subjects . the study subjects were recruited according to the study protocol as reported previously . to identify the low - hdl families , first degree relatives of the probands were examined . the family members were coded as affected if their hdl - c levels were below the 10th percentile cut - off levels . if at least two affected subjects were identified in the family , all available second - degree relatives and spouses were contacted and included in the study . the blood samples for the lipid and lipoprotein analysis were taken at the same visit as the inflammation parameters and therefore the lipid and lipoprotein values in this study represent the values of that visit , and not those of the screening visit . altogether 211 low - hdl family members and 157 normolipidemic subjects had measures of their apoe phenotype , hscrp , and svcam-1 concentrations . the normolipidemic group comprised healthy volunteers and normolipidemic spouses from the low - hdl families . they were required to have no signs of chd or diabetes , and no lipid lowering medication . the smoking status of the patients was categorized into current smokers and nonsmokers ( including exsmokers ) . hypertension was defined as self - reported use of antihypertensive drugs , systolic blood pressure 140 mmhg , or diastolic blood pressure 90 mmhg . estrogen use was defined as any means of estrogen administration ( oral contraceptives , or oral or transdermal postmenopausal hormone replacement therapy ) . serum levels of sicam-1 , svcam-1 , and se - selectin were measured by using commercially available monoclonal antibody - based elisas ( r & d systems , minneapolis , mn , usa ) . serum highly sensitive crp ( hscrp ) was determined by a commercial kit from medix biochemica , kauniainen , finland . statistical comparisons of clinical and biochemical parameters were performed with spss 11.0 for windows ( spss inc . , chigaco , il , usa ) . the frequency distribution between the groups was compared with the chi - square test . the correlation coefficients between the biochemical and clinical characteristics were examined by pearson 's correlation for continuous variables . multiple regression analysis was performed to determine the relative contribution of different variables to hscrp and svcam-1 values . in the low - hdl group , because the subjects in the present cohort were not independent , we used the family number ( which indicates belonging to a certain family ) as a random variable . family - number - adjusted residuals of log crp and log vcam were used in the multiple regression model for low - hdl family members . since control subjects were not related , the family - number adjustment was performed only in the low - hdl group . the effects of apoe phenotype was studied by dividing subjects into two subgroups ( apoe groups ) : into the e3 group , which included e3/2 and e3/3 phenotypes , and into the e4 group which included e4/2 , e4/3 , and e4/4 phenotypes . table 2 shows that serum hscrp values were significantly lower in the e4 than in the e3 subgroup both in the low - hdl group ( p < .01 ) and in the normolipidemic group ( p < .05 ) after adjustment for age , bmi , serum apoa - i , apoa - ii , apob , hdl - c , ldl - c , triglycerides , smoking status , hypertension , and gender in normolipidemic subjects , and after additional adjustment for family number in the low - hdl group . the concentration of serum svcam-1 was significantly lower in the e4 group than in the e3 group in low - hdl subjects ( p < .01 ) but the significance disappeared after adjustment for family number ( table 2 ) . multiple regression analysis revealed that apoe phenotype was a significant predictor of serum hscrp values both in low - hdl and normolipidemic subjects ( p = .023 and p = .011 , resp ) ( table 3 ) . estrogen used was also a strong predictor of serum hscrp concentration in low - hdl subjects ( p = .026 ) as well as in normolpidemic subjects ( p = .036 ) ( table 3 ) the major finding of the present study is that subjects presenting the apoe4 allele have decreased levels of serum hscrp . this is an interesting observation , since apoe plays a central role in inflammation and atherosclerosis . apoe knockout mice are especially prone to atherosclerosis and to die from lipopolysaccharide ( lps)-induced sepsis . apoe participates in cholesterol efflux , and has been observed to be recycled from triglyceride - rich lipoproteins into hdl - particles [ 10 , 26 ] . in mice , exogenous apoe injection has been demonstrated to decrease the lps - induced production of proinflammatory cytokines , probable by decreasing the release of cytokines from macrophages , which was accompanied by reduced mortality . in addition , human crp transgene expression has been shown to cause accelerated atherosclerosis in apolipoprotein e - deficient mice . thus , according to these studies , the role of apoe in inflammation and atherosclerosis may be protective . despite the importance of apoe in the inflammatory process , only few studies have linked apoe polymorphisms to inflammatory parameters . in the central nervous system , apoe4 has been reported to be less effective than apoe3 or apoe2 at suppressing the activation of microglia in cell culture models of brain inflammation . transgenic mice expressing the apoe4 allele have significantly greater systemic and brain elevations of proinflammatory cytokines tnf - alpha and il-6 as compared with their apoe3 counterparts . in the present study , the hscrp concentration was significantly lower in subjects exhibiting apoe4 allele than in those exhibiting other alleles . likewise , mnttri and associates demonstrated , in a population of 179 dyslipidemic finnish men , that subjects with apoe4/3 phenotype had lower mean crp concentration than those with apoe3/3 phenotype . in japanese americans , crp values have been reported to be significantly higher in apoe2 carriers than in apoe3 or apoe4 carriers , and similar between apoe3 and apoe4 carriers . in line with the japanese data , german patients with or without coronary heart disease also showed higher crp values in apoe2 carriers than in apoe3/4 heterozygotes or in apoe4/4 homozygotes . however , crp was also significantly higher in apoe3/3 homozygotes than in apoe3/4 heterozygotes or in apoe4/4 homozygotes . similarly , in the cardiovascular health study , crp values were lower in apoe4 positive than in apoe4 negative individuals independently of the amount of used alcohol . thus , the present data extend these previous observations to a cohort of low - hdl family members as well as to normolipidemic subjects . the mechanisms behind low serum crp values in apoe4 carriers in this study , and in previous works [ 1618 , 31 ] are unclear . since apoe phenotype remained as a predictor of hscrp in multiple regression model , the difference between apoe4 and apoe3 groups can not be explained by confounding factors , such as estrogen use . one explanation , originally suggested by mrz and associates , could be that the metabolism of crp is related to mevalonate pathway . in carriers of apoe4 , the mevalonate pathway may be down - regulated resulting in decreased crp . the observed low serum crp values in apoe4 carriers in the present study , and reportedly low crp values in apoe4 carriers in previous works [ 1618 , 31 ] , are perplexing findings since apoe4 allele is associated to an increased risk of atherosclerosis . however , in the present study , the low - hdl family members within the e4 group had an atherogenic lipid profile : they had higher concentrations of apob , a trend for higher serum triglycerides , and lower concentration of hdl - c as compared with the e3 group . thus , the changes in lipoproteins rather than changes in inflammatory parameters may explain the atherogenity of apoe4 allele . we conclude that apoe phenotype has a strong influence on c - reactive protein . apoe may be a double - faced protein having different actions in lipid metabolism and inflammation . the clinical characteristics and lipid values of the study subjects . data are expressed as means sems . * p < .05 , p < .06 after adjustment for smoking . the concentrations of hscrp , svcam-1 , sicam-1 , and se - selectin in low - hdl subjects and normolipidemic subjects divided by apoe subgroups . data are expressed as means sems . * * p < .01 ; * p < .05 for the comparison with the e3 group after adjustment for age , gender , bmi , hypertension , smoking status , and lipoprotein variables ; and family number in the low - hdl group . multiple regression analysis of predictors of crp values in low - hdl and normolipidemic subjects . 0.202 ( p < .001 ) for low - hdl subjects and 0.326 ( p < .001 ) for normolipidemic subjects when all variables are included . the dependent variable in low - hdl family members is the family number adjusted residual of log crp , and in normolipidemic subjects , the dependent variable is log crp . the aim of this study was to examine the relationship between cartilage , ligament , and meniscus degeneration and radiographic alignment in severe varus knee osteoarthritis in order to understand the development of varus knee osteoarthritis . fifty - three patients ( 71 knees ) with primary varus knee osteoarthritis and who underwent total knee arthroplasty were selected for this study . there were 6 men and 47 women , with 40 right knees and 31 left knees studied ; their mean age at operation was 73.5 years . the ligament , meniscus , degeneration of joint cartilage , and radiographic alignments were examined visually . the tibial plateau tibial shaft angle was larger if the condition of the cartilage in the lateral femoral condyle was worse . the femorotibial angle and tibial plateau tibial shaft angle were larger if the conditions of the lateral meniscus or the cartilage in the lateral tibial plateau were worse . based on the results of this study , progression of varus knee osteoarthritis may occur in the following manner : medial knee osteoarthritis starts in the central portion of the medial tibial plateau , and accompanied by medial meniscal extrusion and anterior cruciate ligament rupture , cartilage degeneration expands from the anterior to the posterior in the medial tibial plateau . bone attrition occurs in the medial tibial plateau , and the femoro - tibial angle and tibial plateau therefore , the lateral intercondylar eminence injures the cartilage of the lateral femoral condyle in the longitudinal fissure type . osteoarthritis is a degenerative condition that increases in incidence and prevalence with age and is a major cause of morbidity in people older than 65 years . this condition most commonly occurs at the knee , particularly in the medial tibiofemoral compartment , and varus knee osteoarthritis accounts for more than 95% of knee osteoarthritis in japan . despite the occurrence of significant morbidity , the etiology and factors involved in the pathogenesis of osteoarthritis and the development of varus knee osteoarthritis have not been fully elucidated . there have been few reports that compared cartilage , ligament , and meniscus degeneration and radiographic alignment in patients with severe varus knee osteoarthritis . the aim of this study was to examine the relationship between cartilage , ligament , and meniscus degeneration and radiographic alignment in patients with severe varus knee osteoarthritis in order to understand the development of varus knee osteoarthritis , observing in detail the degeneration of the knee compartments of the patients who had total knee arthroplasty for their severe varus knee osteoarthritis . over a 4-year period , 53 patients ( 71 knees ) were selected for this study . the inclusion criteria for the study were primary osteoarthritis in a varus knee and total knee arthroplasty in our hospital . no previous history of anterior cruciate ligament ( acl ) damage was found in these patients , and no previous operations had been done in their affected knee joints . there were 6 men and 47 women , with 40 right knees and 31 left knees studied . the mean age at operation was 73.5 years ( range 60 - 86 years ) . the acl and posterior cruciate ligament ( pcl ) were also inspected and divided into 3 groups : intact , ruptured , or disappeared ( table 2 ) . the lateral meniscus ( lm ) and medial meniscus ( mm ) were also inspected and divided into 3 groups : intact , degenerated , and ruptured or disappeared . assessments of the anterior cruciate ligament ( acl ) , posterior cruciate ligament ( pcl ) , and lateral meniscus ( lm ) in severe varus knee osteoarthritis . intact : visually as having an intact synovial sheath , no fiber disruption , and solid insertion points in the femur and tibia , and clinical assessment demonstrated a hard end point . ruptured : the synovial sheath was not present , and fibers were visually attenuated or stretched , and when clinical assessment demonstrated a soft end point . disappeared : the synovial sheath was not present and the fibers were visually absent . degeneration of joint cartilage in the lateral and medial femorotibial joints were also examined visually . using the international cartilage repair society ( icrs ) classification , cartilage degeneration was divided into 5 grades : grade 0 was normal ; grade 1 was superficial lesions that included soft indentation and/or superficial fissures and cracks ; grade 2 was lesions extending down to less than 50% of cartilage depth ; grade 3 was cartilage defects extending down more than 50% of cartilage depth , as well as down to calcified layer and down to but through the subchondral bone ; and grade 4 was exposure of subchondral bone . the femorotibial joint was divided into 4 zones : lateral femoral condyle ( lfc ) , lateral tibial plateau ( ltp ) , medial femoral condyle ( mfc ) , and medial tibial plateau ( mtp ) . cartilage degeneration of the lfc and ltp was divided into 4 types : type i was defined as all areas of the lfc or ltp were grade 0 of the icrs classification ; type ii was defined as most of the degenerated cartilage area in the lfc or ltp was grade1 ; type iii was defined as most of the degenerated cartilage area in the lfc or ltp was grade 2 ; and type iv was defined as most of the degenerated cartilage area in the lfc or lpt was grade 3 or 4 . moreover , the medial compartment of the tibial plateau was subdivided into 3 zones using the icrs cartilage injury evaluation package : anterior , central , and posterior . based on the wear pattern of the mtp , severe varus knee osteoarthritis was divided into 4 types . ( a ) type a is defined as subchondral bone that is exposed only in the central part of the medial tibial plateau ( mtp ) . ( b ) type b is defined as subchondral bone that is exposed in the anterior and central part of the mtp . ( c ) type c is defined as subchondral bone that is exposed in the central and posterior part of the mtp . ( d ) type d is defined as subchondral bone that is exposed from the anterior to the posterior part of the mtp . the femorotibial angle ( fta ) of the lower limb in the frontal plane four angles were then measured to assess the varus deformity of each knee in figure 2 . four angles to assess the varus deformity of each knee on standard preoperative weightbearing anteroposterior x - rays . ( a ) femorotibial angle ( fta ) between the anatomical axis of the femur and that of the tibia . femoral shaft angle ( fcfs ) between the anatomical axis of the femur and the tangent to the subchondral plate of the femoral condyles . ( c ) femoral condylar tibial plateau angle ( fctp ) between the tangents to the subchondral plates of the femoral and tibial condyles . tibial shaft angle ( tpts ) between the tangent to the subchondral plate of the tibia and the anatomical axis of the tibia . the assessments of the acl , pcl , and lm were compared with the radiographic alignment , and cartilage degeneration of the lateral compartment ( lfc and ltp ) was compared with the radiographic alignment . the type of mtp was also compared with the assessments of the acl , pcl and mm , as well as the radiographic alignment . all data are expressed as mean and standard deviation , and statistical software ( spss version 22 , ibm , armonk , ny , usa ) was used for data analysis . statistical analyses were performed using the mann - whitney u test ( 2 groups ) , tukey - kramer test ( more than 2 groups ) , and the test . if the p value was less than 0.05 , the difference was considered significant . the assessments of the acl , pcl , and lm in severe varus knee osteoarthritis are shown in table 2 . the apl normal group was defined as one in which the acl , pcl , and lm were all intact in severe varus knee osteoarthritis , and the apl abnormal group was defined to include all other cases . with regard to each of the parameters of radiographic alignment , only the fta of the apl normal group was significantly smaller than that of the apl abnormal group ( table 3 ) . therefore , the fta of the patients who had normal acl , pcl , and lm in varus knee osteoarthritis was significantly smaller than the others . the apl normal group was defined as the group in which anterior cruciate ligament ( acl ) , posterior cruciate ligament ( pcl ) , and lateral meniscus ( lm ) were all intact in severe varus knee osteoarthritis , and the apl abnormal group was defined to include all other cases . there is a significant difference ( p = 0.0400 , mann - whitney u test ) . the figures in the parentheses indicate the number of cases . the comparison between the cartilage degeneration of the lfc and the radiographic alignment is shown in table 4 . the tibial plateau therefore , if the bone attrition in the medial tibia increased , the cartilage degeneration in the lateral femoral condyle increased . abbreviations : lfc , lateral femoral condyle ; tpts , tibial plateau tibial shaft angle ; fta , femorotibial angle ; ltp , lateral tibial plateau . there is a significant difference between types i and iv ( p = 0.0443 , tukey - kramer test ) . the figures in parentheses indicate the number of cases . there is a significant difference between types i and iv ( p = 0.0331 , tukey - kramer test ) . there are significant differences between types i and iv and between types ii and iv ( p = 0.0101 , tukey - kramer test ) . the figures in the parentheses indicate the number of cases . the comparison between the cartilage degeneration of the ltp and the radiographic alignment is shown in table 4 . the fta was significantly smaller in type i than in type iv , and the tpts was significantly larger in type iv than in types i and ii . there were no significant differences in the other parameters of radiographic alignment in the cartilage degeneration of the ltp . therefore , if the bone attrition in the medial tibia increased , the cartilage degeneration in the lateral tibial plateau increased . the comparison between the assessment of the lateral meniscus and the radiographic alignment is shown in table 5 . the fta was significantly smaller in type i than in type iii , and the tpts was significantly smaller in type i than in types ii and iii . there were no significant differences in the other parameters of radiographic alignment in the assessment of the lm . therefore , if the bone attrition in the medial tibia increased , the degeneration of the lateral meniscus increased . abbreviations : fta , femorotibial angle ; tpts , tibial plateau tibial shaft angle . there is a significant difference between types i and iii ( p = 0.0118 , tukey - kramer test ) . there are significant differences between types i and ii , and between types i and iii ( p = 0.0009 , tukey - kramer test ) . the figures in parentheses indicate the number of cases . there were no significant differences in the age at operation by medial tibial plateau type . the ratio of intact acl or pcl by the type of medial tibial plateau is shown in figure 3 . there was a significant positive correlation between intact acl and type a medial tibial plateau ( test ; p = 0.0096 ) . the ratio of surviving mm , including intact or degenerated , by the type of medial tibial plateau is shown in figure 4 . independent of the type of medial tibial plateau , the middle and posterior segments of the mm disappeared more than the other portion of the mm . the ratio of the intact anterior cruciate ligament ( acl ) or posterior cruciate ligament ( pcl ) by the type of medial tibial plateau ( mtp ) is shown . there is a significant positive correlation between intact acl and type a mtp ( p = 0.0096 , test ) . the ratio of survived medial meniscus , including intact or degenerated , by the type of medial tibial plateau ( mtp ) is shown . independent of the type of mtp , the middle and posterior segments of the medial meniscus ( mm ) disappeared more than the other portion of the mm . ah , anterior horn ; as , anterior segment ; ms , middle segment ; ps , posterior segment ; ph , posterior horn . in the present study , it appeared that , in the medial compartment , cartilage degeneration in the mtp first occurred in the central portion , and there was a significant positive correlation between intact acl and type a mtp . reported that the wear pattern in the mtp was limited mainly to the central portion in intact acl knees and was observed to enlarge into the adjacent zone in acl - deficient knees . because the femur moves on the tibial plateau in a combination of rolling and sliding , and it has a wider contact area compared with the tibia ; tibias presumably sustain constant mechanical stresses and , therefore , reflect the effect of mechanical stresses more than do femurs . biomechanical descriptions of knee flexion and axial rotation during kneeling or squatting are consistent with the more pronounced anteromedial and posterolateral cartilage wear patterns observed in saudi arabian knees . it can be suggested that changes in histomorphometric parameters of subchondral bone are secondary to cartilage damage and proceed deeper into subchondral bone with increasing cartilage degeneration . bone mineral density of the subchondral tibial bone is significantly correlated with future joint space narrowing and could be used as a predictor of knee osteoarthritis progression . the ratio of intact acl in patients with varus osteoarthritis undergoing total knee arthroplasty was reported to be 35.7% or 58.6% ; it was 44% in the present study . the ratio of disappeared acl was reported to be 25% or 7.1% ; it was 15.5% in the present study . acl rupture is not the primary etiology of the cartilage degenerative changes of the knee in patients affected by late arthritis . it seems that acl rupture might be caused by late degenerative changes due to femoral intercondylar notch abrasion and by the increased joint shear force due to the absence of the physiological cartilage layer . forty - six percent of pcls from patients in the osteoarthritis group had moderate or marked degenerative change on light microscopy , but macroscopically , it was 15% in the present study . the enlargement and displacement of the wear pattern in the mtp seems to be worsened by damage or absence of the mm . found that severe cartilage wear in the central portion of the mtp was a median of 20% when the mm was intact , suggesting the occurrence of a protective function of the meniscus , even when the acl was deficient . in the present study , the ratio of the middle segment of the mm was also the smallest in type a mtp . there was a strong correlation between the degree of mm subluxation and the severity of medial joint space narrowing . in the present study , independent of the type of mtp , the middle and posterior segments of the mm disappeared more than the other portion of the mm . hunter et al . reported that cartilage loss was thought to be the major determinant of alignment , and that other factors , including meniscal degeneration and position , bone attrition , osteophytes , and ligament damage , contribute to the variance in malalignment . the knee bone mineral density distribution in the proximal tibia of the 113 patients was negatively correlated with the axial deformity . increasing varus knee alignment was associated with increasing risk of medial compartment joint space narrowing and osteophytes . in the present study , if varus knee osteoarthritis progressed , the fta , especially the tpts , increased significantly . we thought that one of the main causes of progression of varus knee osteoarthritis was bone attrition in the mtp . reported that the sclerotic changes associated with late - stage osteoarthritis were a late manifestation of the disease . sclerotic changes probably reflected a response of the subchondral bone to the increased load bearing and friction that occurred after loss of the articular cartilage . varus thrust was a potent risk factor , identifiable by simple gait observation , for osteoarthritis progression in the medial tibiofemoral compartment . ackroyd reported that the degenerative process usually started in the anterior half of the tibial plateau and in the corresponding part of the medial femoral condyle when the knee was in 20 to 30 of flexion . subregional analysis suggested that , compared with healthy knees , cartilage changes in radiographic osteoarthritis might occur in both directions . based on the results of the present study , the progression of varus knee osteoarthritis may occur in the following manner : medial knee osteoarthritis starts in the central portion of the mtp , and , accompanied by medial meniscal extrusion and acl rupture , cartilage degeneration expands from the anterior to the posterior in the mtp . therefore , the lateral intercondylar eminence results in an injury of the cartilage of the longitudinal fissure type in the lateral femoral condyle , as shown in figure 5 . only the longitudinal fissure is shown in the lateral femoral condylar cartilage . in the comparison of tibial plateau tibial shaft angle ( tpts ) by lateral femoral condyle ( lfc ) type , type i tpts is significantly smaller than type iv . in medial knee osteoarthritis , it appears that the lateral intercondylar eminence first injure the longitudinal fissure in the lateral femoral condylar cartilage .

the study was aimed to compare inflammatory parameters between carriers of apoe4 isoforms ( apoe4/3 , apoe4/2 , and apoe4/4 phenotypes ) and those of carrying apoe3 isoform without apoe4 isoform ( apoe3/3 phenotypes and apoe2/3 phenotypes ) . the concentrations of serum hscrp , svcam-1 , sicam-1 , and se - selectin were measured in 211 subjects from finnish low - hdl families and in 157 normolipidemic subjects . the subjects with apoe4 isoform had lower concentrations of serum hscrp both in low - hdl family members ( p < 0.05 ) and in normolipidemic subjects ( p < 0.01 ) . the differences in serum crp values remained significant after adjustment for age , bmi , smoking status , hypertension , gender , lipoprotein variables , and family number . we conclude that apoe phenotype has a strong influence on serum crp values . objectivesthe aim of this study was to examine the relationship between cartilage , ligament , and meniscus degeneration and radiographic alignment in severe varus knee osteoarthritis in order to understand the development of varus knee osteoarthritis.designfifty-three patients ( 71 knees ) with primary varus knee osteoarthritis and who underwent total knee arthroplasty were selected for this study . there were 6 men and 47 women , with 40 right knees and 31 left knees studied ; their mean age at operation was 73.5 years . the ligament , meniscus , degeneration of joint cartilage , and radiographic alignments were examined visually.resultsthe tibial plateau tibial shaft angle was larger if the condition of the cartilage in the lateral femoral condyle was worse . the femorotibial angle and tibial plateau tibial shaft angle were larger if the conditions of the lateral meniscus or the cartilage in the lateral tibial plateau were worse.conclusionbased on the results of this study , progression of varus knee osteoarthritis may occur in the following manner : medial knee osteoarthritis starts in the central portion of the medial tibial plateau , and accompanied by medial meniscal extrusion and anterior cruciate ligament rupture , cartilage degeneration expands from the anterior to the posterior in the medial tibial plateau . bone attrition occurs in the medial tibial plateau , and the femoro - tibial angle and tibial plateau tibial shaft angle increase . therefore , the lateral intercondylar eminence injures the cartilage of the lateral femoral condyle in the longitudinal fissure type . thereafter , the cartilage degeneration expands in the whole of the knee joints .

in 2011 , the centers for disease control and prevention reported that nearly 26 million american adults , 8.3% of the population , had type 2 diabetes ( 1 ) . the alarming increase in overweight and obesity has played a pivotal role in the rise of type 2 diabetes prevalence . the obesity epidemic has also been associated with an increased prevalence of sleep disturbances , particularly obstructive sleep apnea ( osa ) ( 2,3 ) . over the past decade , both laboratory and epidemiologic studies have identified poor sleep quality and osa as putative novel risk factors for type 2 diabetes ( 46 ) . osa is a treatable chronic sleep disorder characterized by recurrent episodes of complete ( apnea ) or partial ( hypopnea ) obstruction of the upper airway . osa leads to intermittent hypoxemia and hypercapnia , increased oxidative stress , cortical microarousals , sleep fragmentation , and chronic sleep loss . hypopnea index ( ahi ) 5 events per hour , in nondiabetic obese adults have ranged from 50 to 68% ( 7,8 ) . in recent years , evidence has accumulated to indicate that osa is both a risk factor for type 2 diabetes and an exceptionally frequent comorbidity with an adverse impact on glycemic control . five independent studies , totaling nearly 1,400 patients with type 2 diabetes , have shown that the prevalence of osa ( assessed by polysomnography [ psg ] ) ranges between 58 and 86% ( 9,10 ) . independent of adiposity and other known confounders between the presence and severity of osa and insulin resistance and glucose intolerance in nondiabetic adults ( 1114 ) . two studies that used the gold standard of in - laboratory psg to accurately quantify the severity of osa reported a robust association between increasing osa severity and increasing levels of hemoglobin a1c ( hba1c ) in patients with type 2 diabetes , after controlling for multiple potential confounders ( 9,15 ) . while the findings of these two studies suggested that the effective treatment of osa may be a nonpharmacologic strategy to improve glucose control , the results of the only randomized , placebo - controlled clinical trial examining the impact of continuous positive airway pressure ( cpap ) treatment on hba1c in patients with type 2 diabetes were surprisingly disappointing ( 16 ) . one potential reason for the failure of osa treatment to improve chronic glycemic control in patients with type 2 diabetes is insufficient cpap use . notably , the mean nightly cpap use in this clinical trial was 3.6 h. as most of rapid eye movement ( rem ) sleep occurs in the early morning hours before habitual awakening , one possibility is that with suboptimal adherence to cpap therapy , obstructive apneas and hypopneas during rem sleep were disproportionately untreated compared with events in non - rem ( nrem ) sleep . this may be relevant to glycemic control because it is now well established that compared with nrem sleep , rem sleep is associated with greater sympathetic activity in healthy subjects as well as in patients with osa ( 1719 ) . further , compared with events in nrem sleep , obstructive apneas and hypopneas during rem sleep last nearly 30 s longer and are associated with significantly larger oxygen desaturation ( 2022 ) . therefore , obstructive events during rem sleep , as compared with nrem sleep , may have a larger adverse effect on insulin release and action . this issue has major clinical implications for the duration of cpap use that is needed to reverse the negative consequences of osa on glycemic control in type 2 diabetes . we have therefore performed a detailed analysis comparing the contributions of nrem versus rem osa to glycemic control as assessed by levels of hba1c in a large cohort of adults with type 2 diabetes . we prospectively recruited subjects with established type 2 diabetes using an advertisement posted in the primary care and endocrinology clinics at the university of chicago , inviting participation in a research study on sleep and diabetes . eligible individuals had to meet the criteria for type 2 diabetes based on physician diagnosis using standard criteria ( 23 ) . in order to include individuals with newly diagnosed type 2 diabetes , we also recruited in the community using an advertisement inviting subjects at risk for type 2 diabetes based on age and adiposity to participate in a research study on sleep and metabolism . all participants without an established diagnosis of type 2 diabetes had to undergo a standard 75-g oral glucose tolerance test and meet the american diabetes association guidelines for the diagnosis of type 2 diabetes ( 23 ) . all participants were in stable condition and , when on pharmacological treatment , on a stable antidiabetic medication regimen for the preceding 3 months . exclusion criteria were unstable cardiopulmonary conditions , neurological disorders , psychiatric disease , shift work , chronic insomnia , or any prior or current treatment for osa ( upper airway surgery , cpap therapy , oral appliances , or supplemental oxygen ) . we previously reported the association of osa severity categories ( no , mild , moderate , and severe osa ) with chronic glycemic control in type 2 diabetes using 60 of the participants included in this analysis ( 9 ) . the study was approved by the university of chicago institutional review board , and all participants gave written informed consent . subjects were admitted to the clinical resource center or the sleep research laboratory of the sleep , health , and metabolism center at the university of chicago to undergo an overnight in - laboratory psg . self - reported ethnicity - based diabetes risk was categorized as low - risk category ( non - hispanic whites ) and high - risk category ( african americans , hispanics , and asians ) . the duration of type 2 diabetes and the number of medications were verified by questionnaires as well as review of the patients medical records . hba1c values ( defined as the proportion of hemoglobin that is glycosylated ) were obtained from the patient s chart if assessed during the previous three months ( n = 17,15% of subjects ) or measured on a single blood sample drawn on the morning after the psg ( n = 98 , 85% of the subjects ) . hba1c was measured by bio - rad variant classic boronate affinity - automated high - performance liquid chromatography ( bio - rad , hercules , ca ) . the intra - assay coefficient of variation was 0.51.0% , and the interassay coefficient of variation was 2.22.4% . bedtimes were from 11:00 p.m.12:00 a.m. until 7:00 a.m.8:00 a.m. each subject was recorded for a minimum of 7 h to determine the presence and severity of obstructive respiratory events across the entire night . psg ( neurofax eeg 1100 system , nihon kohden , foothill ranch , ca ) included recordings of six electroencephalographic channels , bilateral electro - oculograms , chin and tibialis electromyogram , electrocardiogram , airflow by nasal pressure transducer and oronasal thermocouples , chest and abdominal wall motion by piezo electrode belts , and oxygen saturation by finger pulse oximeter . all psgs were staged and scored according to the 2007 american academy of sleep medicine manual for the scoring of sleep and related events ( 24 ) . apneas were defined as total cessation of airflow for at least 10 s ( obstructive if respiratory effort was present and central if respiratory effort was absent ) . hypopneas were scored if the magnitude of the ventilation signal decreased by at least 50% of the baseline amplitude of the nasal pressure transducer for at least 10 s and were associated with either a 3% or greater drop in oxygen saturation as measured by finger pulse oximetry or an electroencephalographic microarousal ( 24 ) . the total ahi was defined as the number of obstructive apneas and obstructive hypopneas per hour of sleep . given the minimal presence of central apneas , we did not include these events in the calculation of ahi . the median central apnea index was 0.001 ( interquartile range of 0.0010.41 ) , and the highest central apnea index was 5 . a subject was considered to have mild osa if the ahi was 514 , moderate osa if the ahi was 1529 , and severe osa if the ahi was 30 . rem ahi was calculated as the number of apneas and hypopneas during rem sleep divided by total time in rem sleep in hours . nrem ahi was calculated by dividing the number of apneas and hypopneas during nrem sleep by total time in nrem sleep in hours . the oxygen desaturation index ( odi ) was defined as the total number of oxygen desaturations of at least 3% per total sleep time ( tst ) in hours . the microarousal index ( mai ) was calculated as the total number of microarousals per hour of sleep . differences between subjects with and without osa were tested using the student t test or mann whitney nonparametric test for continuous variables . categorical variables were reported as proportions and were compared using the square test or fisher s exact test . five multivariate linear models were successively fitted to examine associations between hba1c and measures of osa severity after controlling for multiple covariates . model 1 included demographic variables traditionally associated with glycemic control , namely , age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use . lastly , model 5 included all the covariates in model 1 plus nrem ahi and rem ahi . since there are individuals who have a significant number of apneas and hypopneas during rem sleep while having an overall ahi below 5 ( hence no osa based on current definitions ) , we included all 115 subjects ( with or without osa ) in the multivariate regression models in order to explore the entire spectrum of rem and nrem events ( ahi , odi , and mai ) . we formally ruled out any evidence of collinearity among the variables entered in the models using standard statistics , including tolerance and variance inflation factor ( spss statistics v20 , ibm , armonk , ny ) . values for hba1c , years of type 2 diabetes , and rem and nrem ahi were submitted to natural log ( ln ) transformation . in order to deal with zero values , the total ahi , rem ahi , and nrem ahi were log transformed using the formula ahi = log(ahi + 0.01 ) . only one outlier was identified ( low hba1c ) , and sensitivity analysis excluding this subject was performed confirming the association between hba1c and measures of rem osa severity . goodness of fit of the models was assessed using diagnostic plots . in order to estimate the effect size of increasing severity of osa on hba1c in a clinically useful manner , models were fitted to estimate the change in adjusted hba1c based on quartiles of rem and nrem ahi . the models included all the covariates in model 5 ( age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use ) and replaced lnrem ahi with rem ahi quartiles while keeping lnnrem ahi in the model . this process was repeated , and lnnrem ahi was replaced with nrem ahi quartiles while keeping lnrem ahi in the model . similar models were fitted for rem and nrem odi and mai quartiles . to simulate the impact of various durations of nocturnal cpap therapy on hba1c , we calculated the mean profiles of cumulative minutes of rem and nrem sleep over 8 h of total recording time from the 115 polysomnograms . we then estimated mean percentages of rem and nrem sleep left untreated after 4 , 6 , and 7 h of optimal cpap treatment eliminating all events . for each duration of cpap use , we entered the number of rem and nrem obstructive events left untreated in a regression model predicting hba1c after adjustment for age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use . all statistical analyses were performed using spss statistics v20 and verified using stata ( v10.1 , college station , tx ) . we prospectively recruited subjects with established type 2 diabetes using an advertisement posted in the primary care and endocrinology clinics at the university of chicago , inviting participation in a research study on sleep and diabetes . eligible individuals had to meet the criteria for type 2 diabetes based on physician diagnosis using standard criteria ( 23 ) . in order to include individuals with newly diagnosed type 2 diabetes , we also recruited in the community using an advertisement inviting subjects at risk for type 2 diabetes based on age and adiposity to participate in a research study on sleep and metabolism . all participants without an established diagnosis of type 2 diabetes had to undergo a standard 75-g oral glucose tolerance test and meet the american diabetes association guidelines for the diagnosis of type 2 diabetes ( 23 ) . all participants were in stable condition and , when on pharmacological treatment , on a stable antidiabetic medication regimen for the preceding 3 months . exclusion criteria were unstable cardiopulmonary conditions , neurological disorders , psychiatric disease , shift work , chronic insomnia , or any prior or current treatment for osa ( upper airway surgery , cpap therapy , oral appliances , or supplemental oxygen ) . we previously reported the association of osa severity categories ( no , mild , moderate , and severe osa ) with chronic glycemic control in type 2 diabetes using 60 of the participants included in this analysis ( 9 ) . the study was approved by the university of chicago institutional review board , and all participants gave written informed consent . subjects were admitted to the clinical resource center or the sleep research laboratory of the sleep , health , and metabolism center at the university of chicago to undergo an overnight in - laboratory psg . self - reported ethnicity - based diabetes risk was categorized as low - risk category ( non - hispanic whites ) and high - risk category ( african americans , hispanics , and asians ) . the duration of type 2 diabetes and the number of medications were verified by questionnaires as well as review of the patients medical records . hba1c values ( defined as the proportion of hemoglobin that is glycosylated ) were obtained from the patient s chart if assessed during the previous three months ( n = 17,15% of subjects ) or measured on a single blood sample drawn on the morning after the psg ( n = 98 , 85% of the subjects ) . hba1c was measured by bio - rad variant classic boronate affinity - automated high - performance liquid chromatography ( bio - rad , hercules , ca ) . the intra - assay coefficient of variation was 0.51.0% , and the interassay coefficient of variation was 2.22.4% . bedtimes were from 11:00 p.m.12:00 a.m. until 7:00 a.m.8:00 a.m. each subject was recorded for a minimum of 7 h to determine the presence and severity of obstructive respiratory events across the entire night . psg ( neurofax eeg 1100 system , nihon kohden , foothill ranch , ca ) included recordings of six electroencephalographic channels , bilateral electro - oculograms , chin and tibialis electromyogram , electrocardiogram , airflow by nasal pressure transducer and oronasal thermocouples , chest and abdominal wall motion by piezo electrode belts , and oxygen saturation by finger pulse oximeter . all psgs were staged and scored according to the 2007 american academy of sleep medicine manual for the scoring of sleep and related events ( 24 ) . apneas were defined as total cessation of airflow for at least 10 s ( obstructive if respiratory effort was present and central if respiratory effort was absent ) . hypopneas were scored if the magnitude of the ventilation signal decreased by at least 50% of the baseline amplitude of the nasal pressure transducer for at least 10 s and were associated with either a 3% or greater drop in oxygen saturation as measured by finger pulse oximetry or an electroencephalographic microarousal ( 24 ) . the total ahi was defined as the number of obstructive apneas and obstructive hypopneas per hour of sleep . given the minimal presence of central apneas , we did not include these events in the calculation of ahi . the median central apnea index was 0.001 ( interquartile range of 0.0010.41 ) , and the highest central apnea index was 5 . a subject was considered to have mild osa if the ahi was 514 , moderate osa if the ahi was 1529 , and severe osa if the ahi was 30 . rem ahi was calculated as the number of apneas and hypopneas during rem sleep divided by total time in rem sleep in hours . nrem ahi was calculated by dividing the number of apneas and hypopneas during nrem sleep by total time in nrem sleep in hours . the oxygen desaturation index ( odi ) was defined as the total number of oxygen desaturations of at least 3% per total sleep time ( tst ) in hours . the microarousal index ( mai ) differences between subjects with and without osa were tested using the student t test or mann whitney nonparametric test for continuous variables . categorical variables were reported as proportions and were compared using the square test or fisher s exact test . five multivariate linear models were successively fitted to examine associations between hba1c and measures of osa severity after controlling for multiple covariates . model 1 included demographic variables traditionally associated with glycemic control , namely , age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use . lastly , model 5 included all the covariates in model 1 plus nrem ahi and rem ahi . since there are individuals who have a significant number of apneas and hypopneas during rem sleep while having an overall ahi below 5 ( hence no osa based on current definitions ) , we included all 115 subjects ( with or without osa ) in the multivariate regression models in order to explore the entire spectrum of rem and nrem events ( ahi , odi , and mai ) . we formally ruled out any evidence of collinearity among the variables entered in the models using standard statistics , including tolerance and variance inflation factor . values for hba1c , years of type 2 diabetes , and rem and nrem ahi were submitted to natural log ( ln ) transformation . in order to deal with zero values , the total ahi , rem ahi , and nrem ahi were log transformed using the formula ahi = log(ahi + 0.01 ) . only one outlier was identified ( low hba1c ) , and sensitivity analysis excluding this subject was performed confirming the association between hba1c and measures of rem osa severity . goodness of fit of the models was assessed using diagnostic plots . in order to estimate the effect size of increasing severity of osa on hba1c in a clinically useful manner , models were fitted to estimate the change in adjusted hba1c based on quartiles of rem and nrem ahi . the models included all the covariates in model 5 ( age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use ) and replaced lnrem ahi with rem ahi quartiles while keeping lnnrem ahi in the model . this process was repeated , and lnnrem ahi was replaced with nrem ahi quartiles while keeping lnrem ahi in the model . similar models were fitted for rem and nrem odi and mai quartiles . to simulate the impact of various durations of nocturnal cpap therapy on hba1c , we calculated the mean profiles of cumulative minutes of rem and nrem sleep over 8 h of total recording time from the 115 polysomnograms . we then estimated mean percentages of rem and nrem sleep left untreated after 4 , 6 , and 7 h of optimal cpap treatment eliminating all events . for each duration of cpap use , we entered the number of rem and nrem obstructive events left untreated in a regression model predicting hba1c after adjustment for age , sex , ethnicity - based diabetes risk , bmi , years of type 2 diabetes , and insulin use . all statistical analyses were performed using spss statistics v20 and verified using stata ( v10.1 , college station , tx ) . therefore , the study was completed by 131 participants . those who obtained less than 4 h of tst during the psg were not included in the analysis ( n = 7 ) . participants were also excluded if the psg data could not be interpreted due to multiple artifacts in the airflow signal ( n = 8) . one patient showed severe oxygen desaturation not explained by apneas or hypopneas consistent with significant hypoventilation . of the 115 subjects included in the study , 56.5% were women , 58.3% were african american , 68.7% were obese , and the mean bmi was 34.5 kg / m . the median duration of type 2 diabetes was 4 years , and a quarter of the subjects were not on any antidiabetic medication . i.e. , neuropathy , nephropathy , retinopathy , coronary artery disease , or peripheral arterial disease ) . mild osa was present in 27% , moderate osa in 28.7% , and severe osa in 29.6% of the subjects . there were no significant differences in sex , race , bmi , years of type 2 diabetes , number of antidiabetic medications , insulin use , and hba1c level between subjects with and without osa , but participants without osa were on average 9 years younger than those with osa . the lack of statistically significant differences in bmi and hba1c may have been related to the small number of subjects without osa . there were no significant differences in total recording time and percentage of slow wave sleep between subjects with and without osa . however , subjects with osa had significantly less tst and percentage of rem sleep and significantly higher wake after sleep onset . within the participants with osa ( n = 98 ) , rem ahi , rem odi , and rem mai were all significantly higher than nrem ahi , nrem odi , and nrem mai . the odi was more than fourfold higher during rem than nrem sleep , but differences in mai were more modest . demographic and clinical features of 115 patients with type 2 diabetes table 2 describes the results of the five multivariate linear regression models predicting hba1c . model 3 shows that nrem ahi was not associated with hba1c ( p = 0.070 ) . in contrast , in model 4 , rem ahi was independently associated with hba1c ( p = 0.001 ) . in model 5 , rem ahi ( p = 0.008 ) remained a significant predictor of hba1c even after adjusting for nrem ahi ( p = 0.762 ) . in the final fully adjusted model 5 , the independent predictors of increased hba1c were rem ahi ( p = 0.008 ) , race risk ( p = 0.001 ) , years of type 2 diabetes ( p = 0.001 ) , and insulin use ( p < 0.001 ) . similar results were obtained when nrem ahi and rem ahi were replaced by the total number of events in nrem and rem sleep , respectively ( p = 0.023 for rem events and p = 0.355 for nrem events ) . multivariate linear regression models predicting natural log of hba1c in patients with type 2 diabetes in order to estimate the effect size of increasing levels of rem ahi and nrem ahi on hba1c , we performed multivariate linear regression models using quartiles of rem ahi and nrem ahi . 1 , after adjustment for age , sex , bmi , race risk , years of type 2 diabetes , insulin use , and lnnrem ahi , increasing quartiles of rem ahi were significantly associated with increasing levels of hba1c ( p = 0.044 for linear trend ) . the mean adjusted hba1c increased from 6.3% in subjects with rem ahi < 12.3 events per hour ( lowest quartile ) to 7.3% in subjects with rem ahi > 47 events per hour ( highest quartile ) . similarly , quartiles of rem odi and rem mai were significantly associated with increasing levels of hba1c . the mean adjusted hba1c increased from 6.5% in the lowest quartile of rem odi to 7.5% in the highest quartile ( p = 0.039 for linear trend ) . similarly , the mean adjusted hba1c increased from 7.6% in the lowest quartile of rem mai to 8.9% in the highest quartile ( p = 0.003 for linear trend ) . in contrast , increasing levels of nrem ahi , nrem odi , and nrem mai quartiles were not associated with hba1c ( fig . 1 ) . adjusted mean hba1c values for rem and nrem ahi , odi , and mai quartiles . for all the panels , multivariate linear regression models were fitted to estimate the mean natural ln hba1c adjusted for demographic variables traditionally associated with glycemic control such as age , sex , ethnicity - based diabetes risk , bmi , ln years of type 2 diabetes , and insulin use . in addition , panels are adjusted for ( a ) lnnrem ahi , ( b ) lnrem ahi , ( c ) lnnrem odi , ( d ) lnrem odi , ( e ) lnnrem mai , and ( f ) lnrem mai . age and bmi are centered at their means : 55 years old and 35 kg / m , respectively . the corresponding -coefficients for each quartile were then exponentiated to convert from ln hba1c to the standard values of hba1c . figure 2a illustrates the predominance of rem sleep in the later part of sleep . in our cohort , 3 and 4 h after lights off , on average only 25 and 40% of rem sleep had occurred , respectively . optimally titrated cpap use for 3 or 4 h would treat only 25 or 40% of rem sleep , respectively , and would leave most obstructive events during rem sleep untreated . figure 2b and c illustrate the simulated impact of 4 , 6 , and 7 h of cpap use in men and women with low and high race / ethnicity - based diabetes risk . this simulation clearly shows that the metabolic benefit of 4 h of cpap use , often considered as adequate cpap compliance , is modest , while a much more clinically significant effect can be obtained when treatment is extended to 6 h and beyond . cumulative minutes of rem and nrem sleep over 8 h of bedtime ( a ) and simulation of various hours of cpap use in men and women with type 2 diabetes based on race / ethnicity - based diabetes risk ( b and c ) . a : data are summarized as mean sd of cumulative rem and nrem sleep minutes from lights off to lights on in 115 subjects with type 2 diabetes . the mean duration of rem and nrem sleep in our cohort was 82 and 298 min , respectively . using cpap for 3 or 4 h from the time lights are turned off will cover only 25 or 40% of rem sleep , respectively , and will leave most obstructive events during rem sleep untreated . b and c : simulation of the impact of 4 , 6 , and 7 h of cpap use in four groups of subjects based on sex and race / ethnicity - related diabetes risk . with this simulation , 4 h of cpap use would treat 40% of rem sleep and would lead to a drop in adjusted hba1c of 0.230.28% . in contrast , 7 h of cpap therapy would treat 87% of rem sleep and lead to a decrease in adjusted hba1c between 0.87 and 1.1% . this study reveals that hba1c , a measure of chronic glycemic control in patients with type 2 diabetes , is adversely associated with obstructive apneas and hypopneas that occur in rem sleep ( rem ahi ) but not in nrem sleep ( nrem ahi ) . the independent association between rem ahi , rem odi , and rem mai and hba1c is robust and of clinical significance , with a difference of 1.0% hba1c between the lowest and highest quartiles of rem ahi as well as rem odi and of 1.3% hba1c between the lowest and highest quartiles of rem mai after adjusting for all the covariates . these effect sizes are comparable to what would be expected from widely used antidiabetic medications . the severity of osa in our cohort was greater in rem sleep than in nrem sleep , as evidenced by a higher ahi and a nearly fourfold higher odi . thus , despite the shorter duration of rem sleep , exposure to the adverse consequences of osa , particularly intermittent hypoxemia , was greater during rem than nrem sleep . surprisingly , in our diabetic participants with osa , nrem ahi only predicted approximately 25% of the variance of rem ahi . whether hyperglycemia plays a role in this relative independence of the severity of rem osa relative to nrem osa remains to be determined . multiple mechanistic pathways are likely to be involved in the link between rem osa and poorer glycemic control in subjects with type 2 diabetes . when compared with nrem sleep or quiet wakefulness , rem sleep is associated with increased sympathetic activation and reduced vagal tone in normal subjects and even more so in patients with osa ( 1719 ) . most endocrine organs releasing hormones involved in glucose regulation are sensitive to changes in sympathovagal balance . well - documented examples relevant to metabolic risk are pancreatic insulin secretion , hepatic glucose production , and adipocyte regulation of energy balance ( 2527 ) . in addition , peptidergic factors originating from the intestine ( glucagon - like peptide-1 and glucose - dependent insulinotropic polypeptide ) augment the insulin response induced by nutrients . the secretion of these incretin hormones is intimately linked to autonomous nervous system ( 2830 ) . however , it is important to point out that the impact of osa on sympathetic activation in patients with type 2 diabetes of long duration remains unclear and that long - standing hyperglycemia may lead to reduction in sympathetic activity . lastly , obstructive apneas and hypopneas during rem sleep lead to greater degrees of hypoxemia than in nrem sleep ( 21,22 ) . in the present cohort , rem odi was indeed much greater than nrem odi . intermittent hypoxemia has been shown to be toxic to -cell function in murine models of sleep apnea ( 31,32 ) . the findings from our analyses strongly suggest that rem - related obstructive respiratory events are of clinical significance for the severity of type 2 diabetes . two recent studies that performed continuous interstitial glucose monitoring simultaneously with psg directly support our hypothesis that rem - related osa may have adverse metabolic consequences ( 33,34 ) . one of these studies included 13 obese patients with type 2 diabetes with severe osa and compared them with 13 obese patients with type 2 diabetes without osa with similar demographic characteristics . although there was no difference in the mean diurnal glycemic level between the two groups , the mean glycemic level was 38% higher during rem sleep in those with osa ( 33 ) . they found that in the absence of osa , rem sleep leads to a larger decline in interstitial glucose concentration than nrem sleep . in contrast , osa during nrem sleep had no impact on interstitial glucose concentrations ( 34 ) . taken together , the evidence from studies assessing interstitial glucose levels supports our finding that obstructive events during rem sleep are adversely associated with glucose metabolism . while our participants did not meet any proposed definition of rem - related or rem - predominant osa ( 35,36 ) , our findings suggest that failure to recognize and treat osa in rem sleep may be of critical clinical significance for glycemic control in diabetic patients . in clinical practice , 4 h of nightly cpap use is considered adequate adherence to therapy ( 37 ) . indeed , a randomized controlled trial of cpap therapy in patients with type 2 diabetes reported an average use of 3.6 h per night ( 16 ) . the severity of residual osa was not estimated . the disappointing results of cpap trial in type 2 diabetes may reflect the failure to treat rem osa due to insufficient cpap use , leaving most obstructive events during rem sleep untreated . alternatively , there may be other factors beyond poor cpap adherence that led to a lack of improvement in glycemic control such as a poor reserve in -cell function . our analyses show that based on the distribution of cumulative rem sleep in our cohort , cpap therapy for the first 4 h after lights off would leave 60% of obstructive events during rem sleep untreated and would be associated with a decrease in the adjusted hba1c by only 0.250.28% . in contrast , 7 h of optimal cpap therapy would be associated with a decrease in the adjusted hba1c by 0.871.1% . first , we used hba1c , the most commonly used measure in clinical practice , to assess glycemic control . therefore , we can not ascertain whether the mediating pathways linking rem ahi to hba1c involve increased insulin resistance or impaired -cell function . moreover , we only measured hba1c at a single time point , which was not consistently on the same day as psg . however , treatment was stable for the preceding 3 months in all participants , and hba1c was measured on the morning after the psg in 98 out of 115 participants ( 85% ) . although hba1c reflects glycemic control 10 to 12 weeks before the assay , it mostly reflects glucose fluctuations during the last 6 weeks of the measurement . despite our efforts to ensure treatment stability in the prior 3 months , we can not exclude the possibility that fluctuations in adherence to medications may have influenced hba1c levels . our study did not assess associations between rem osa and glucose control in subjects with prediabetes or normal glucose tolerance . we also had a large proportion of african americans and subjects requiring little or no antidiabetic medications . therefore , it would be important for our findings to be replicated in larger and more diverse cohorts , including participants with more diabetes complications and/or longer disease duration as well as individuals with prediabetes or with normal glucose tolerance but at high risk for type 2 diabetes . also , we did not have a measure of habitual sleep duration , which may be important in evaluating chronic exposure to rem osa , and our only measure of adiposity was the bmi . lastly , the cross - sectional nature of the study does not address the direction of causality . indeed , only rigorously designed intervention studies will provide causal evidence between disordered breathing during rem sleep and glucose metabolism dysregulation . in summary , our findings support the notion that osa in rem sleep has a strong and clinically significant association with glycemic control in subjects with type 2 diabetes . since rem sleep is dominant during the latter part of the sleep period , rem - related osa may often remain untreated with 4 h of cpap use . our analyses suggest that to achieve clinically significant improvement in glycemic control in patients with type 2 diabetes , cpap use may need to be extended beyond 6 h per night . further research is needed to elucidate the mechanistic pathways linking osa during rem sleep and adverse metabolic outcomes . peripheral veno - arterial extracorporeal membrane oxygenation ( va - ecmo ) is increasingly used in acute , severe cardiogenic shock refractory to conventional therapy . although percutaneous femoral insertion of va - ecmo is straightforward , the procedure is usually performed after intubation under general anaesthesia , and invasive ventilation is continued for the whole period of extracorporeal support . here , we show that va - ecmo can be successfully performed with neither ( non- ) invasive ventilation nor deep analgosedation . a 51-year - old man with a history of hypertension and diabetes mellitus presented to the local hospital in cardiogenic shock ( blood pressure , 80/40 mmhg ) . electrocardiography ( ecg ) revealed a ( sub)acute anterior myocardial infarction complicated by complete atrioventricular block and a slow ventricular escape rhythm of 30 beats per minute ( fig . 1 ) . after initial medical therapy ( volume resuscitation , inotropes ) , the clinical condition allowed urgent transport to an interventional centre . on arrival , immediate transvenous ventricular pacing echocardiography showed preserved wall thickness , but extensive apical and anteroseptal akinesia and inferior hypokinesia , with a large left ventricular ( lv ) apical thrombus ( left ventricular ejection fraction ( lvef ) , 10 % ) . 1serial electrocardiograms ( upper panel ) and transthoracic echocardiograms ( lower panel ) on presentation ( left ) and on recovery 6 weeks later ( right ) showing a large dilated anterior myocardial infarction with partial recovery of contractile function . arrow indicates lv apical thrombus , which dissolved over time after therapeutic anticoagulation ( recovery ) . rv right ventricle , lv left ventricle serial electrocardiograms ( upper panel ) and transthoracic echocardiograms ( lower panel ) on presentation ( left ) and on recovery 6 weeks later ( right ) showing a large dilated anterior myocardial infarction with partial recovery of contractile function . arrow indicates lv apical thrombus , which dissolved over time after therapeutic anticoagulation ( recovery ) . rv right ventricle , lv left ventricle since cardiogenic shock persisted , va - ecmo was initiated under local anaesthesia via femoral access ( 25f venous cannula , 19f arterial cannula ) as a bridge to recovery and additional therapy . during the procedure , the patient was fully awake , non - intubated and breathing oxygen through a non - rebreathing facemask . va - ecmo yielded 4-l / min flow , and a mean arterial pressure of 6070 mmhg was achieved . an adequately supported circulation was reflected by normal blood gas analysis , lactate levels , venous oxygen saturation and renal function ; sinus rhythm ensued on day 2 . coronary angiography revealed severe three - vessel disease ( proximally occluded right , significantly stenosed circumflex and subtotally occluded left anterior descending coronary artery ) unattractive for immediate revascularisation . the clinical course was complicated by obvious signs of pulmonary oedema on serial chest radiographs ( fig . 2 ) in the absence of dyspnoea ( respiratory rate of 1520 per minute ) and with adequate oxygenation ( sao2 , 97100 % ; pao2 > 100 mmhg ) . therefore , heart failure therapy ( captopril , eplerenone , isosorbide mononitrate ) was tailored with a pulmonary artery catheter ( pac ) and intensified ( furosemide , hydrochlorothiazide ) to accomplish negative fluid balances . daily gradual tapering of ecmo flow ( l / min ) during weaning off ecmo support ( lower panel ) serial chest x - rays indicating decrease of pulmonary congestion ( upper panel ) . daily gradual tapering of ecmo flow ( l / min ) during weaning off ecmo support ( lower panel ) after 4 days of va - ecmo under low - dose continued intravenous analgesia ( 1 mg morphine / h ) , support flow was gradually reduced ( 1 l / min per day ) under therapeutic heparinisation and pac monitoring . on day 8 , echocardiography revealed partial lv recovery ( lvef , 25 % ) during normally conducted sinus rhythm ( fig . 1 ) and 1 l / min support flow , allowing weaning off the va - ecmo . in a stable condition , the patient was mobilised and discharged from the intensive care unit ( icu ) on day 11 . based on myocardial viability studies ( mid anteroseptal viability on cardiac magnetic resonance perfusion imaging ) , a drug - eluting stent was placed in the left anterior descending artery on day 21 , followed by clinical discharge on day 37 . there is accumulating evidence that severe , refractory cardiogenic shock can be supported by percutaneous va - ecmo as a bridge to recovery , long - term mechanical support and eventually heart transplantation . yet , va - ecmo is cumbersome , as patients are generally managed and instrumented in the icu with invasive ventilation , analgosedation and feeding via a nasogastric tube . this renders va - ecmo a complex and invasive therapeutic approach with numerous potential icu - related complications . in contrast to va - ecmo standard care , we managed this case without intubation , ventilation or significant analgosedation , which has , to the best of our knowledge , not been reported in this way before . we used pac monitoring for haemodynamic tailoring and strived for optimal filling pressures ( wedge pressures < 15 mmhg ) , while allowing adequate systemic va - ecmo flow . during va - ecmo weaning , we monitored cardiac recovery by continuously measuring intrinsic cardiac output on gradual daily reduction of support flow ( fig . 1 ) moreover , spontaneous respiration was carefully monitored by pulse oximetry and repeated blood gas analyses , while pulmonary oedema was aggressively treated by combined diuretic therapy ( fig . 1 ) . this strategy seems imperative as , for example , pneumonia may render va - ecmo without intubation impossible . others have reported on awake va - ecmo requiring additional percutaneous atrioseptostomy to treat pulmonary congestion , which was not necessary in our patient . moreover , neurological performance remained unremarkable and could easily be evaluated in this cooperative patient . neurological assessment is of utmost importance during peripheral va - ecmo , especially on recovering cardiac function and pending pulmonary oedema , which might cause hypoxaemia of the proximal aortic arch , that is , the harlequin syndrome . after roughly a week of va - ecmo support , cardiac recovery was accompanied by immediate physical mobilisation creating optimal conditions for the subsequent clinical course . va - ecmo for severe and refractory cardiogenic shock may successfully be performed without mechanical ventilation . careful monitoring of volume status and respiratory condition is imperative and allows keeping patients comfortable and cooperative also during prolonged periods of extracorporeal support . the limited invasiveness of this stand - alone va - ecmo approach may encourage a more widespread and accessible use in cardiogenic shock .

objectiveseverity of obstructive sleep apnea ( osa ) has been associated with poorer glycemic control in type 2 diabetes . it is not known whether obstructive events during rapid eye movement ( rem ) sleep have a different metabolic impact compared with those during non - rem ( nrem ) sleep . treatment of osa is often limited to the first half of the night , when nrem rather than rem sleep predominates . we aimed to quantify the impact of osa in rem versus nrem sleep on hemoglobin a1c ( hba1c ) in subjects with type 2 diabetes.research design and methodsall participants underwent polysomnography , and glycemic control was assessed by hba1c.resultsour analytic cohort included 115 subjects ( 65 women ; age 55.2 9.8 years ; bmi 34.5 7.5 kg / m2 ) . in a multivariate linear regression model , rem apnea hypopnea index ( ahi ) was independently associated with increasing levels of hba1c ( p = 0.008 ) . in contrast , nrem ahi was not associated with hba1c ( p = 0.762 ) . the mean adjusted hba1c increased from 6.3% in subjects in the lowest quartile of rem ahi to 7.3% in subjects in the highest quartile of rem ahi ( p = 0.044 for linear trend ) . our model predicts that 4 h of continuous positive airway pressure ( cpap ) use would leave 60% of rem sleep untreated and would be associated with a decrease in hba1c by approximately 0.25% . in contrast , 7 h of cpap use would cover more than 85% of rem sleep and would be associated with a decrease in hba1c by as much as 1%.conclusionsin type 2 diabetes , osa during rem sleep may influence long - term glycemic control . the metabolic benefits of cpap therapy may not be achieved with the typical adherence of 4 h per night . we report on the use of percutaneous femoral veno - arterial extracorporeal membrane oxygenation ( va - ecmo ) in a fully awake , non - intubated and spontaneously breathing patient suffering from acute , severe and refractory cardiogenic shock due to a ( sub)acute anterior myocardial infarction . intensified heart failure therapy was closely monitored with a pulmonary artery catheter and allowed gradual weaning off the ecmo support without additional invasive measures , notably without mechanical ventilation . neurological assessment was possible at all times and complete physical mobilisation was straightforward directly after weaning from ecmo . this limited invasive approach may encourage a more widespread use of percutaneous va - ecmo .

gas storage in porous materials is a desirable technology that has been significantly developed in recent years , owing to its potential to address numerous persisting challenges in a number of industrial applications related to energy , environment , and health care sectors . in the context of clean energy , there is an amplified willingness to reduce greenhouse gas emissions , caused by energy production processes , as evidenced by the considerable ongoing research in academia and industry alike aiming to develop practical solutions to mitigate this problem . correspondingly , appropriate studies have been conducted in order to practically deploy relatively cleaner alternative fuels such as methane ( ch4 ) , a primary component of natural gas ( ng ) and biogas . ch4 is of great interest as a fuel for stationary and mobile applications due to ( i ) its high h to c ratio in comparison to other fossil fuels , resulting in relatively lower co and co2 emissions , and ( ii ) its lower sulfur and nitrogen contents , leading to lessened sox and nox emissions . the aforementioned attributes position ch4 as a fuel appreciably cleaner than gasoline and diesel . nonetheless , the main drawback of ch4 , in comparison to liquid fossil fuels , is its low volumetric energy density . therefore , the development of suitable and sustainable on - board vehicle methane storage solutions , close to room temperature , is vital to the successful deployment of methane as a conventional fuel for transport applications . highly porous materials represent an interesting category of adsorbents that display distinct structural advantages for ch4 storage . the appropriate combination of a high surface area associated with a considerable pore volume with a suitable pore shape and functionality , in a given porous material , is crucial to achieve the desired enhanced ch4 storage uptake and a practical working capacity at a set pressure and temperature . it is notable that the volumetric working capacity is an essential parameter to assess the material s performance toward ch4 storage . the working capacity represents the usable amount of ch4 derived by subtracting the unused adsorbed ch4 , corresponding to the uptake at the delivery pressure ( 5 bar ) , from the uptake at the maximum adsorption pressure ( 35 bar or higher ) . prominently , one of the pathways to enhance the methane working capacity of a given porous material is to regulate its methane uptake at relatively low pressures and subsequently reduce the unused ch4 uptake up to the 5 bar threshold . relatedly , the storage of other gases such as nitric oxide ( no ) and carbon dioxide ( co2 ) has been previously studied and explored for various relevant applications . in contrast , studies pertaining to high - pressure oxygen ( o2 ) storage are still scarce . the availability of high amounts of o2 is of prime importance in the health care domain , particularly in the treatment of respiratory insufficiencies and in hyperbaric oxygen changes for the treatment of carbon monoxide poisoning . correspondingly , a large amount of oxygen is used to enrich air during catalyst regeneration in the catalytic cracking units . markedly , there is a significant need to develop efficient pathways to store o2 for various industrial needs . metal organic frameworks ( mofs ) , a special class of solid - state materials , have emerged as modular and functional porous materials that can offer potential to address many enduring challenges pertaining to energy and environmental sustainability . principally , advances in mof chemistry have permitted the successful implementation of reticular chemistry ; predesigned building blocks were directed to assemble into a preset network topology . namely , the molecular building block ( mbb ) approach has been deployed to construct targeted functional mofs , where desired structural and geometrical attributes are incorporated into the mbbs prior to the assembly process . in effect , isolating reaction conditions that consistently permit in situ formation of the requisite inorganic mbbs , in the presence of a suitable organic linker , is essential for the successful implementation of the mbb approach and the subsequent formation of the targeted mof with the desired network topology . advantageously , the mbb approach permits the rational assembly of targeted mofs and their subsequent structural fine tuning using isoreticular chemistry . the deliberate alteration of a mof dimensionality and functionality without changing its underlying topology , isoreticular chemistry , is regarded as a powerful pathway for the development of new functional materials with distinctive properties . evidently , the selection of an appropriate mof platform , with desired topological attributes for the logical practice of isoreticular chemistry , offers the potential to readily access porous materials suitable to address the ongoing gas storage challenges . practically , several key requisites have been considered and targeted in order to facilitate the attainment of a high - storage mof media : ( i ) inorganic mbbs based on light and abundant elements , ( ii ) organic mbbs amenable to size , shape , and functionality adjustments via expansion and decoration , ( iii ) an elect mof platform that permits access to isoreticular mofs with concomitant high surface area , large pore volume , and fine - tuned pores in the micropore domain , and ( iv ) an mof platform based on an underlying topology that prohibits interpenetration upon mof expansion . in this context , considering the aforementioned requisites , we identified the mof platform based on the soc topology ( square - octahedral ) as a promising platform to access isoreticular mof materials for potential use in gas storage and separation applications . the soc - mof platform offers interesting structural features where the pore system comprised of cavities and channels can be fine tuned in the micropore domain by the judiciously fine tuning the square building unit ( the tetracarboxylate ligand ) . it is notable that the first reported soc - mof materials ( in- , ga- , and fe - based soc - mof ) revealed an exceptionally high gas storage density for h2 and ch4 despite their associated moderate surface area and pore volume in comparison to the best storage mof materials . congruently , we found it compelling to target soc - mofs with relatively larger surface areas and pore volumes via isoreticular chemistry , where the expansion strategy is employed to construct isoreticular soc - mofs based on selected / compatible and expanded organic mbbs , and subsequently evaluate their performance for storage of valuable commodities such as ch4 , h2 , co2 , and o2 . markedly , the construction of a highly microporous mof with a soc topology requires the judicious selection of an expanded rectangular organic linker that facilitates the in situ formation of the targeted inorganic oxo - centered trinuclear m(iii ) cluster [ m3(3-o)(o2c)6 ] ( m = in , al , fe , etc . ) . specifically , targeting mofs based on the trinuclear aluminum(iii ) cluster will permit the development of a relatively low cost material with tailored properties for gas storage applications . it is important to note that mofs based on the trinuclear al(iii ) cluster [ al3(3-o)(o2c)6 ] , a 6-connected mbb , are scarce , with only a few examples reported in the literature due to challenges in isolating reaction conditions that allow the in situ formation of the aforementioned inorganic mbb . in this work , we report the synthesis and the structure , on the basis of single - crystal x - ray diffraction ( scxrd ) studies , of the first aluminum mof having the soc topology and possessing an exceptional porosity . importantly , this is the first report disclosing the assembly of the oxo - centered trinuclear aluminum(iii ) cluster ( oxo - centered aluminum(iii ) trimer ) [ al3(3-o)(o2c)6 ] , with a quadrangular ligand into a given mof , namely the highly microporous al - soc - mof-1 with more than 2 cm / g pore volume and 6000 m / g apparent langmuir surface area . furthermore , the use of similar reaction conditions , which afforded the synthesis of the parent al - soc - mof-1 , in the presence of functionalized tetracarboxylate linkers ( with naphthalene or anthracene replacing the phenyl core in the parent ligand ) resulted in two new isoreticular structures : namely the naphthalene species al - soc - mof-2 and the anthracene species al - soc - mof-3 . extensive gas adsorption studies were carried out on these isoreticular soc - mofs with different gases ( n2 , co2 , ch4 , o2 ) at low pressures ( cryogenic temperatures ) and at high pressures . in particular , ch4 and o2 adsorption isotherms were investigated experimentally at different temperatures and over a wide range of pressures up to 85 and 120 bar , respectively . it was found that al - soc - mof-1 has one of the highest ever total and working gravimetric ch4 uptakes at 35 bar and higher pressures at any given temperature . in contrast to all other best mofs reported to date in the open literature for ch4 storage , the parent al - soc - mof-1 sorption studies revealed an enhancement in the volumetric ch4 storage working capacity when the temperature was decreased . particularly , at 258 k and 80 bar , the al - soc - mof-1 fulfilled the department of energy ( doe ) target ( both gravimetric and volumetric ) and exhibited the highest working volumetric capacity of 264 cm ( stp)/cm . correspondingly , to the best of our knowledge , al - soc - mof-1 showed the highest gravimetric total capacity for co2 and o2 among microporous mofs . furthermore , molecular simulation studies supported and confirmed our experimental results for ch4 storage and thus encouraged us to explore various plausible theoretical isoreticular al - soc - mofs , based on expanded ( or contracted ) and/or functionalized tetracarboxylate organic building blocks , for ch4 storage . this study permitted us to ( i ) pinpoint various prospective al - soc - mofs with outstanding ch4 storage capabilities similar to those of the parent al - soc - mof-1 , confirming the superior volumetric and gravimetric storage capabilities of the parent al - soc - mof-1 , and ( ii ) gain a better understanding of the structure property relationship , deriving a better correlation between the soc - mof gas storage properties and the makeup of the soc - mof porous system ( pore shape and size , ligand dimensions and functionalities ) . in our effort to isolate the first aluminum - based soc - mof , numerous attempts have been carried out to isolate reaction conditions that consistently allow the in situ formation of the desired trinuclear aluminum(iii ) mbb [ al3(3-o)(o2c)6 ] . accordingly , we designed and synthesized the tetratopic ligand 3,3,5,5-tetrakis(4-carboxyphenyl)-p - terphenyl ( h4tcpt ) ( h4l1 ) that can act as a rectangular mbb . successfully , reactions between h4l1 and alcl36h2o in acidic solution containing a mixture of n , n-dimethylformamide ( dmf ) and acetonitrile ( ch3cn ) afforded colorless homogeneous crystals with a cube - shaped morphology , characterized and formulated by scxrd as [ al3o(tcpt)1.5(h2o)3]|cl| ( 1 ) . the crystal structure of 1 reveals a 3-periodic framework built up from 3-oxo - centered trinuclear al(iii ) inorganic mbbs [ al3(3-o)(h2o)3(o2c)6 ] . each aluminum cation ( al ) displays an octahedral coordination environment and coordinates to six oxygen atoms : namely , four bis - monodentate deprotonated carboxylate oxygen atoms from four independent tcpt ligands and one 3-oxo anion , and a terminal aqua ligand to complete the coordination sphere . the trinuclear al(iii ) mbbs are bridged by six independent tcpt ligands , resulting in the formation of a 3-periodic cationic framework , al - soc - mof-1 ( figure 1 ) . the charge balance is provided by the presence of chloride ions , which was confirmed by an x - ray photoelectron spectroscopy ( xps ) experiment ( figure s11 in the supporting information ) . crystallographic analysis affirms that the chloride ions are disordered over six positions around the trinuclear al(iii ) cluster with equal probability . this analysis was also supported by a al solid - state nmr spectroscopy experiment ( figure s12 in the supporting information ) . crystal structure of 1 showing the assembly of the trinuclear aluminum(iii ) mbb [ al3(3-o)(h2o)3(o2c)6 ] with the organic ligand h4tcpt ( h4l1 ) ( middle ) and topological analysis of 1 , where the 6-connected trinuclear al(iii ) mbb can be viewed as a trigonal - prismatic sbu , while the organic ligand can be rationalized as a 4-connected building unit to give ( 4,6)-c soc - net ( left ) or can be viewed as 3-c sbus resulting in a ( 3,6)-c derived net edq ( right ) . topological analysis reveals that 1 has the anticipated edge transitive ( 4,6)-connected net with the soc underlying topology . the trinuclear al(iii ) mbb [ al3(3-o)(o2c)6 ] can be regarded as a trigonal - prismatic secondary building unit ( sbu ) with the six points of extension corresponding to the carbon of the carboxylate moieties matching the vertex figure of the 6-c node in the soc net . the 6-c inorganic mbbs are joined by the rectangular organic ligand , 4-c node , into a primitive cubic system arrangement ( figure 1 ) . alternatively from a topological perspective , the 4-c rectangular ligand can be regarded as comprised of two interconnected 3-c triangular sbus that are further linked through the 6-c trigonal - prismatic sbus to afford a mof related to a ( 3,6)-c derived net edq , with transitivity 2 2 ( figure 1 and figures s25 and s26 in the supporting information ) . in this paper , the reported al - mofs will be referred to as al - soc - mofs . in order to isolate other isoreticular analogues of al - soc - mof-1 , the phenyl ring located at the core of the h4tcpt ligand was substituted by 1,4-naphthalenyl and 9,10-anthracenyl cores to give the naphthalene- and anthracene - functionalized ligands 3,3,5,5-tetrakis(4-carboxyphenyl)-1,4-diphenylnaphthalene ( h4tcdpn ) ( h4l2 ) and 3,3,5,5-tetrakis(4-carboxyphenyl)-9,10-diphenylanthracene ( h4tcdpa ) ( h4l3 ) , respectively ( figure 2a ) . as anticipated , under reaction conditions similar to those used to isolate the al - soc - mof-1 , cube - shaped crystals were obtained and characterized using scxrd and powder x - ray diffraction ( pxrd ) studies ( figure 2b ) , revealing the construction of two isoreticular al - soc - mof compounds , naphthalene al - soc - mof-2 ( 2 ) and anthracene al - soc - mof-3 ( 3 ) with the formula [ al3o(ligand)1.5(h2o)3]|cl| . ( a ) representation of the organic mbbs used to construct isoreticular al - soc - mofs . ( c ) nitrogen isotherms at 77 k for the isoreticular al - soc - mofs . the phase purity of each al - soc - mof compound ( 13 ) was confirmed by whole profile pattern matching using the le bail method ( figures s13s15 in the supporting information ) . the al - soc - mof-1 structure encloses cubic - shaped cages 14.3 in diameter delimited by six tcpt ligands , which occupy the faces of the cage , and eight inorganic trinuclear al(iii ) clusters located on the vertices of the cuboidal cage . the cage is accessible through apertures of approximately 5.6 8.4 , taking van der waals ( vdw ) radii into consideration . the structure also encloses two well - defined 1d infinite channels with estimated dimensions of 14 ( vdw ) , which is approximately at the border of microporous materials ( figures s23 and s24 in the supporting information ) . the corresponding solvent - accessible free volumes for 13 were estimated to be 80.5% , 79% , and 75% , respectively , by summing voxels more than 1.2 away from the framework using platon software . in light of the extraordinarily pure microporous architecture exhibited by 13 , optimization of the conventional activation conditions ( drying under vacuum and heating ) showed that the guest solvent in the pores could be easily removed using a traditional approach ( vacuum and heating ) without altering their microporosity . nitrogen ( n2 ) adsorption measurements at 77 k were carried out on the acetonitrile - exchanged samples , showing a fully reversible type i isotherm representative of porous materials with permanent microporosity ( figure 2c ) . g for 2 , and 5212 and 4849 cm / g for 3 ( table 1 ) . it is notable that the resultant high microporosity ( surface area and pore volume ) is exceptional and has not been observed , prior to this work , using a traditional activation method that often causes pore collapse in the case of highly porous mofs . such a unique feature is of prime importance for the implementation and deployment of 13 as gas storage media for onboard or stationary gas storage applications . abet and alang are the experimental bet and langmuir specific surface areas . pvtheo and pvexp are the calculated pore volume from crystal structures and the experimentally measured pore volume , respectively . the successful use of the conventional activation method was confirmed by the excellent agreement between the experimental and the optimal theoretical pore volumes ( pvexp = 2.3 , 2.1 , and 1.8 cm / g and pvtheo = 2.3 , 2.2 , and 1.9 cm / g for 13 , respectively ) . furthermore , al - soc - mof structures preserved their optimal porosity after heating up to 340 c under vacuum ( figures s27b , s34b , and s36b in the supporting information ) , another essential feature that is rarely observed for highly porous mofs . the high thermal stability was also confirmed using variable - temperature pxrd studies and thermal gravimetric analysis ( figures s17s22 in the supporting information ) . interestingly , the extremely open structure , exclusively concerted in the microporous range , combined with the distinctive structural features ( presence of cages and channels ) place al - soc - mofs as ideal adsorbent candidates for gas storage studies . accordingly , ch4 adsorption on 13 was extensively studied experimentally at variable temperatures and up to 80 bar as well as at low pressure and 112 k ( boiling point of ch4 ) . the methane adsorption isotherms at 112 k for 13 revealed remarkable ch4 uptakes near saturation pressures e.g. , 1336 , 1205 , and 1055 cm ( stp)/g at p / p0 = 0.95 , respectively ( figures s28a , s35a , and s37a in the supporting information ) . importantly , high - pressure ch4 adsorption isotherms at variable temperatures , depicted in figure 3 , showed that 1 has one of the highest ch4 gravimetric uptakes ever reported ( ca . 361 cm ( stp)/g ) for any microporous mof materials under the disclosed doe operational storage conditions ( 298 k and 35 bar ) . mesoporous mof-210 and dut-49 displayed uptakes of around 210 and 364 cm(stp)/g under the same conditions . interestingly , the doe ch4 gravimetric uptake target of 700 cm ( stp)/g ( 0.5 g / g ) was addressed and reached for relatively high pressures at temperatures below 288 k : e.g. 50 bar at 258 k and 85 bar at 288 k ( figure 3 ) . single - component gas adsorption isotherms for ch4 at different temperatures for al - soc - mof-1 , showing total ch4 gravimetric uptakes surpassing the doe target at particular pressures and temperatures . additionally , analysis of the volumetric ch4 adsorption isotherms , using the al - soc - mof-1 crystal density , revealed an enhancement in the volumetric ch4 storage working capacity when the temperature was decreased ( figure 4 ) . specifically , the volumetric ch4 storage working capacity for 1 increased from 201 cm ( stp)/cm to 264 cm ( stp)/cm when the temperature was decreased from 298 to 258 k at working pressures between 80 bar ( adsorption ) and 5 bar ( desorption ) . this attribute , unique to al - soc - mof-1 , is unprecedented , as all available ch4 storage data for mofs have shown a conventional decrease in the volumetric ch4 storage working capacity with a decrease in temperature , as illustrated in figure 4 and table s3 in the supporting information for utsa-76 , hkust-1 , ni - mof-74 , nu-111 , and pcn-14 . it is notable that mof-519 was not included in this comparative assessment , as the associated data at low temperatures were not available for this highly ch4 adsorbing mof . comparison of the ch4 volumetric working capacities ( 580 and 565 bar ) at different temperatures ( 258 , 273 , and 298 k ) for al - soc - mof-1 with the best microporous mof materials reported to date . a comprehensive comparison of absolute ch4 uptakes and working capacities for al - soc - mof-1 ( 1 ) with the various best mof materials reported so far under different temperature and pressure conditions is presented in figure 5 and figure s38 in the supporting information . interestingly , although the total volumetric ch4 uptake for 1 is relatively lower than those of some of the highly adsorbing mofs , 1 displayed mutually high volumetric and gravimetric working capacities at different working temperatures and pressures . this notable and rare compromise between the gravimetric and the volumetric capacities for 1 is a result of the reduced unused ch4 uptake below 5 bar and the linear trend of the ch4 isotherms at relatively high pressures , a desirable attribute for an appropriate gas storage medium . in contrast to the best mofs reported so far for ch4 storage , 1 exhibits a relatively low ch4 heat of adsorption ( 11 kj / mol at low loading ) in the relatively low ch4 loading region , only slightly higher than the ch4 latent heat of evaporation ( figure s30 in the supporting information ) . markedly , the comparatively favorable methane adsorption at relatively high pressures can be attributed to enhanced ch4ch4 interactions regulated by the appropriate pore size of al - soc - mof-1 . therefore , the combination of both aforementioned effects , governing the ch4 adsorption at low and high pressures , in a single material afforded the exceptional ch4 working capacities observed for the al - soc - mof-1 , especially at relatively low temperatures . it is notable that 1 exhibits the second highest ch4 volumetric working capacity at 298 k and 580 bar working pressure range : namely , 201 cm ( stp)/cm vs 230 cm ( stp)/cm for the recently reported mof-519 . noticeably , the two synthesized isoreticular al - soc - mofs ( 2 and 3 ) also exhibited high gravimetric and volumetric total and working ch4 uptakes that were only slightly lower than the uptake values derived for 1 . the experimental results for 2 and 3 are summarized in figure s41 and table s4 in the supporting information . total ( a d ) at 65 and 80 bar and 565 and 580 bar working ( e h ) ch4 gravimetric and volumetric uptakes for al - soc - mof-1 ( 1 ) in comparison to the best mof materials reported to date at 298 , 270 , and 240 k. the data for al - soc - mof-1 ( 1 ) were collected at 298 , 273 , and 258 k. with the aim of gaining a better understanding of the structure property relationship governing the resultant high methane storage capacities in the al - soc - mofs , we assessed theoretically the plausible ch4 storage capacity of various plausible theoretical isoreticular al - soc - mofs based on expanded ( or contracted ) and/or functionalized tetracarboxylate organic building blocks . in order to derive a better correlation between the soc - mof gas storage properties and the makeup of the soc - mof porous system ( pore shape and size , ligand dimensions and functionalities ) , a comprehensive molecular simulation study was carried out on various hypothetical / isoreticular al - soc - mofs constructed using hypothetical organic building blocks . first , we envisioned validating our molecular simulation methodology on the parent 1 ( al - soc - mof-1 ) and corroborating the mechanism of ch4 adsorption at 5 bar and at higher pressures , resulting in the outstanding ch4 working capacity . second , we simulated and evaluated the absolute and working ch4 uptakes for various hypothetical isoreticular soc - mofs , constructed using different optimized ( i ) elongated , ( ii ) functionalized , and ( iii ) contracted organic mbbs . in effect , the simulated isoreticular al - soc - mofs were assembled using both experimentally synthesized and hypothetically designed linkers obtained by modifying the arms and/or the core of the al - soc - mof-1 ligand as shown in figure 6 and figure s47 and scheme s2 in the supporting information . figure 6 depicts the naming scheme employed to label the hypothetical al - soc - mof linkers . for example , the original linker is shown in figure 6 ( left ) , where p - p denotes phenyl phenyl : both the arm and the core have one phenyl group . phenyl for the arm and acetylene phenyl acetylene for the core , as shown in figure 6 ( right ) . a total of 18 theoretical analogues were hypothetically assembled , and their associated ch4 adsorption isotherms were simulated . for clarity the new simulated al - soc - mof structures will be named and referred to using the associated linker name . scheme illustrating the adapted naming for the employed hypothetical organic ligands and associated hypothetical al - soc - mofs . initially , grand canonical monte carlo ( gcmc ) simulations of methane adsorption were performed for the parent compound 1 in order to compare first the resulting simulated results with available experimental adsorption data , and subsequently validate the simulation approach adopted in this study ( figure 7a and figure s44 in the supporting information ) . more details about the employed simulation method are described in the supporting information . as shown , the theoretical ch4 adsorption isotherm for p - p is in a good agreement with the experimental data ( 1 ) . the corresponding screenshots of methane adsorption at different relevant pressures , i.e. 5 bar ( limiting desorption pressure ) and 35 , 65 , and 80 bar ( storage pressures ) , are shown in figure 7b . the relatively very low simulated unused methane uptake at 5 bar ( both gravimetric and volumetric ) for p - p equivalent to 1 , at 5 bar , was confirmed by performing adsorption isotherms at different temperatures : namely , at 298 , 295 , 273 , and 258 k ( figure s45 in the supporting information ) . this is reflected in the observed nonpreferential positions of ch4 molecules in the framework of p - p ( figure 7b ) : i.e. , no specific preferential adsorption sites at 5 bar . ( a ) methane sorption in al - soc - mof-1 at 298 k : simulation ( red filled circles ) vs experiment ( black filled squares ) . ( b ) screenshots of methane adsorption in p - p ( equivalent to al - soc - mof-1 ) at 298 k at different pressures : 5 , 35 , 65 , and 80 bar . the purple spheres surrounding the framework represent the methane molecules . delightfully , the simulated ch4 adsorption isotherms on the optimized structure , on the basis of a molecular mechanics simulation approach ( details in the supporting information ) , of the parent 1 ( p - p ) are in good agreement with the corresponding ch4 adsorption isotherms simulated on the experimental structure ( figure s46 in the supporting information ) . accordingly , the same molecular mechanics optimization procedure was employed to construct 18 hypothetical isoreticular al - soc - mofs and subsequently simulate their associated total and working ch4 uptakes using the gcmc approach . the simulated absolute volumetric and gravimetric ch4 adsorption isotherms for p - p ( 1 ) and the other isoreticular al - soc - mofs were simulated at various temperatures and up to 80 bar total pressure ( figures s76s84 in the supporting information ) . interestingly , these results showed that the use of ( i ) elongated , ( ii ) functionalized , and ( iii ) contracted linkers resulted in three distinguished al - soc - mof groups in terms of gravimetric volumetric uptake tradeoff . in fact , the use of ( i ) elongated arms and/or cores , such as pp - pp , led to an increase in the gravimetric uptake at the expense of the volumetric uptake under any pressure and temperature conditions evaluated in this study . ( ii ) functionalizing the phenyl core of the linker led generally to lower gravimetric uptake but still with good gravimetric volumetric uptake tradeoff under any pressure and temperature conditions explored here . in a particular case , strategies for functionalization of the phenyl cores with different functional groups , such as cf3 and br , led to relatively good working volumetric uptakes ( due to the relatively higher framework density ) , albeit with much lower working gravimetric uptakes . interestingly , ( iii ) contraction of the arms and/or the core , for instance the a - p structure , offer potential for a collective improvement ( ideal compromise ) of the gravimetric and volumetric uptakes in comparison to 1 at any temperature , particularly at intermediate pressures of 35 bar ( figure 8) . principally , in terms of ch4 working ( 535 bar ) volumetric and gravimetric capacity tradeoff , contraction of the arms showed a notable improvement in the absolute ch4 uptake at 298 k. this unique compromise can be attributed to the enhanced ch4 adsorption uptake at relatively high pressures below 35 bar , due to the reduced channel dimensions in the a - p structure in comparison to the parent al - soc - mof-1 ( 9.3 10.0 vs 14.0 14.2 ) . it is important to note that the al - soc - mof-1 ( p - p ) still exhibits both enhanced volumetric and gravimetric tradeoff for 565 and 580 bar working pressures at any temperature ( figure 8) . total ( a ) at 35 bar and ( b ) 535 bar working ch4 gravimetric and volumetric uptakes for p - p and a - p simulated structures in comparison to the best mof materials reported to date at 298 k. theoretical total ( left , c and e ) and working ( right , d and f ) gravimetric vs volumetric capacities for selected hypothetical isoreticular al - soc - mofs under a wide range of pressures ( 35 , 65 , and 80 bar ) at different temperatures ( 298 and 258 k ) in comparison to al - soc - mof ( 1 ) . the purple area represents the desired range of the best compromise between gravimetric and volumetric total and working uptakes . a summary of the theoretical results in terms of ch4 absolute and working capacities , volumetric and gravimetric , at 298 , 273 , and 258 k under a wide range of pressures are presented in figures s48s66 in the supporting information . the exceptional methane storage capabilities of al - soc - mof-1 have inspired us to extend this study to other important commodities , namely o2 and co2 . accordingly , we recorded various o2 and co2 adsorption isotherms for al - soc - mof-1 . notably , the amounts of o2 and co2 adsorbed in al - soc - mof-1 near saturation pressures ( 0.95 p / p0 ) , derived from adsorption isotherms at 90.2 and 195.15 k for o2 and co2 , respectively , were found to be remarkably high ( 1757 and 1236 cm ( stp)/g , respectively ) ( figure s29 in the supporting information ) . markedly , the combination of experimentally accessible low pressure ( at 90.2 k ) and high - pressure o2 adsorption data up to 115 bar ( at 298 k ) ( combined with the toth model ) revealed that al - soc - mof-1 exhibits a record of 29 mmol / g absolute gravimetric o2 uptake at 140 bar , which is much higher than those of hkust-1 ( 13.2 mmol / g ) and nu-125 ( 17.4 mmol / g ) , reference materials for this application ( figure 9 ) . additionally , al - soc - mof-1 displayed a record deliverable capacity between 5 and 140 bar of 27.5 mmol / g , vs 11.8 and 15.4 mmol / g for hkust-1 and nu-125 , respectively ( figure 9a ) . consequently , by neglecting the effect of packing density and the void space occupied by the material , a 1 l cylinder filled with al - soc - mof-1 will potentially enhance the volumetric o2 storage capacity ( 172 cm / cm ) by 70% at 100 bar , in comparison to a conventional empty cylinder ( figure 9b ) . it is notable that , if we assume a prospective 25% loss associated with packing density , the al - soc - mof-1 volumetric o2 storage capacity still offers a 25% enhancement over an empty cylinder . analysis of the o2 adsorption recorded at variable temperatures indicated that 1 exhibits a relatively low o2 heat of adsorption ( 10 kj / mol at low loading ) over the whole o2 loading range , slightly higher than the o2 latent heat of evaporation ( figure s33 in the supporting information ) . ( a ) single - component gravimetric gas adsorption isotherm for o2 at 298 k showing that 1 exhibits the highest deliverable uptake reported so far . ( b ) volumetric o2 adsorption isotherm compared to the storage capacity in a pressurized container . additionally , the co2 adsorption studies revealed that al - soc - mof-1 exhibits an exceptional absolute gravimetric co2 uptake at 40 bar of 2 g / g ( 1020 cm ( stp)/g ) vs 1.5 g / g for mof-177 , setting a new record among microporous mofs ( figure s39 in the supporting information ) . consequently , al - soc - mof-1 displays the uppermost working capacity between 1 and 40 bar of 1.90 g / g ( 967 cm ( stp)/g ) vs 1.46 g / g ( 742 cm ( stp)/g ) for mof-177 . an analysis of variable - temperature co2 adsorption data showed that al - soc - mof-1 exhibits a relatively low co2 heat of adsorption ( 17 kj / mol at low loading ) over the whole co2 loading range ( figure s31b in the supporting information ) . it is important to note that the mesoporous mof-210 exhibits the highest absolute gravimetric co2 uptake at 50 bar ( 2.8 g / g ) . finally , it is worth noting that al - soc - mof-1 also exhibits an excellent h2 storage capacity at 77 k ( ca . 11 wt % ) ( figure s40 in the supporting information ) at high pressure ( 30 bar ) in comparison to other highly porous materials . similarly , o2 and co2 adsorption studies were performed for the two isoreticular al - soc - mofs ( 2 and 3 ) , which revealed that the naphthalene and anthracene analogues exhibit only slightly lower o2 and co2 adsorption uptakes under the same conditions in comparison to 1 ( figures s42 and s43 and table s4 in the supporting information ) . in summary , we successfully employed the molecular building block approach to synthesize the first aluminum soc - mof isoreticular materials . specifically , reaction conditions that consistently permit the in situ generation of the [ al3(3-o)(h2o)3(o2c)6 ] mbb were isolated and used for the construction of a highly porous ( 4,6)-connected aluminum based soc - mof , al - soc - mof-1 , with more than 6000 m / g langmuir specific surface area . importantly , tedious activation using dry supercritical co2 is not required in order to activate the al - soc - mof-1 and its naphthalene and anthracene analogues . in particular , the conventional activation technique , i.e. a simple combination of heating and vacuum ( or n2 flush ) , is sufficient for full activation prior to gas loading unloading cycles . extensive gas adsorption studies were carried out on the al - soc - mof platform with different gases ( n2 , co2 , ch4 , o2 , etc . ) . methane adsorption isotherms were completed at different temperatures and over a wide range of pressures , up to 85 bar . interestingly , it was found that al - soc - mof-1 exhibits one of the highest total and working gravimetric ch4 uptakes at 35 bar . in contrast to the other reported best mofs for ch4 storage , al - soc - mof-1 showed enhanced ch4 storage working capacity as the temperature was decreased . particularly , at 258 k and 80 bar , al - soc - mof-1 fulfils the doe target and exhibits the highest working volumetric capacity of 264 cm ( stp)/cm . to the best of our knowledge , this is the first time that a porous material has fulfilled both the challenging gravimetric and volumetric targets for the ch4 working capacity . the collective experimental and gcmc simulation studies indicated that the parent al - soc - mof-1 , in contrast to various hypothetical isoreticular al - soc - mofs based on contracted , elongated , and functionalized ligands , exhibits the best compromise between the volumetric and gravimetric total and working uptakes over a wide range of pressure and temperature conditions . details on the synthesis of the organic ligands used in this study , 3,3,5,5-tetrakis(4-carboxyphenyl)-p - terphenyl ( h4tcpt ) , 3,3,5,5-tetrakis(4-carboxyphenyl)-1,4-diphenylnaphthalene ( h4tcdpn ) , and 3,3,5,5-tetrakis(4-carboxyphenyl)-9,10-diphenylanthracene ( h4tcdpa ) , are provided in the supporting information . single - crystal diffraction data were collected at beamline i19 , diamond light source , didcot , u.k . , using the wavelength 1.0402 at 250 k. fourier - transform infrared ( ft - ir ) spectra ( 4000600 cm ) were collected in the solid state on a nicolet 700 ft - ir spectrometer . the peak intensities are described in each of the spectra as very strong ( vs ) , strong ( s ) , medium ( m ) , weak ( w ) , broad ( br ) , and shoulder ( sh ) . powder x - ray diffraction ( pxrd ) measurements were performed on a panalytical mpd xpert pro x - ray diffractometer at 45 kv and 40 ma for cu k ( 1.5418 ) equipped with a variable - temperature stage , with a scan speed of 20/min . the sample was held at the designated temperature for at least 10 min between each scan . high - resolution dynamic thermal gravimetric analysis ( tga ) was performed under a continuous n2 flow and recorded on a ta instruments high - resolution tgaq500 thermogravimetric analyzer . low - pressure gas sorption measurements were performed on a fully automated autosorb-1c gas sorption analyzer ( quantachrome instruments ) . high - pressure gas sorption studies were performed with a magnetic suspension balance marketed by rubotherm ( bochum , germany ) . a solution of alcl36h2o ( 29 mg , 0.015 mmol ) and h4l1 ( 7.1 mg , 0.01 mmol ) in n , n - dimethylformamide ( dmf ) ( 1 ml ) , acetonitrile ( ch3cn ) ( 1 ml ) , and nitric acid ( 3.5 m , 0.5 ml ) was prepared in a 20 ml scintillation vial and subsequently placed into a preheated oven at 130 c for 12 h to give pure small colorless cube - shaped crystals . suitable single crystals were obtained using the same synthetic procedure , but with an increase in the amount of hno3 to l ml . crystals of al - soc - mof-1 were harvested , washed with ch3cn , and air - dried . ft - ir ( 4000650 cm ) : 3349 ( br ) , 1605 ( s ) , 1592 ( s ) , 1423 ( s ) , 1387 ( vs ) , 1312 ( w ) , 1243 ( w ) , 1100 ( w ) , 1018 ( w ) , 854 ( w ) , 830 ( w ) , 783 ( s ) , 771 ( s ) , 701 ( s ) . a solution of alcl36h2o ( 29 mg , 0.015 mmol ) and h4l2 ( 7.6 mg , 0.01 mmol ) in n , n - dimethylformamide ( dmf ) ( 1 ml ) , acetonitrile ( ch3cn ) ( 1 ml ) , and nitric acid ( 3.5 m , 0.5 ml ) was prepared in a 20 ml scintillation vial and subsequently placed into a preheated oven at 130 c for 12 h to give pure small colorless cube - shaped crystals . suitable single crystals were obtained using the same synthetic procedure , but with an increas in the amount of hno3 to l ml . crystals of al - soc - mof-2 were harvested , washed with ch3cn , and air - dried . ft - ir ( 4000650 cm ) : 3349 ( br ) , 1606 ( s ) , 1545 ( m ) , 1422 ( s ) , 1384 ( s ) , 1241 ( w ) , 1100 ( w ) , 1015 ( w ) , 851 ( w ) , 851 ( w ) , 771 ( s ) , 705 ( m ) . a solution of alcl36h2o ( 29 mg , 0.015 mmol ) and h4l3 ( 8.1 mg , 0.01 mmol ) in n , n - dimethylformamide ( dmf ) ( 1 ml ) , acetonitrile ( ch3cn ) ( 1 ml ) , and nitric acid ( 3.5 m , 0.3 ml ) was prepared in a 20 ml scintillation vial and subsequently placed into a preheated oven at 130 c for 12 h to give a pure microcrystalline yellow powder . suitable single crystals were obtained using the same synthetic procedure , but with an increase in the amount of hno3 to 1 ml crystals of al - soc - mof-3 were harvested , washed with ch3cn , and air - dried . ft - ir ( 4000650 cm ) : 3349 ( br ) , 1606 ( s ) , 1547 ( s ) , 1442 ( s ) , 1387 ( s ) , 1312 ( w ) , 1241 ( w ) , 1181 ( w ) , 1016 ( w ) , 852 ( m ) , 771 ( s ) , 706 ( s ) . each year , an estimated 10 million health care office visits to gynecologists are due to vulvovaginitis . vulvovaginitis refers to a variety of inflammatory lower genital tract disorders that may be secondary to infection , irritation , allergy , or systemic disease . infectious causes of vulvovaginitis include bacterial vaginosis , candidiasis , and trichimoniasis ; while noninfectious causes include exposure to chemicals , allergens , genital atrophy , and trauma . of the infectious vaginitides , only vulvovaginitis due to candida offers over the counter medications for women to self diagnose and treat their condition . these nonprescription antifungals , introduced to the market in 1990 , are among the top ten best selling over - the - counter drugs in the us with annual sales of approximately $ 250 million . one survey reports that 73% of women with recurrent vulvovaginitis have resorted to over - the - counter medications to reduce health care cost and avoid an expensive office visit . further , homeopathic drug sales were estimated at $ 201 million in 1995 and have steadily risen to an estimated 300450 million in 2003 [ 3 , 4 ] . this article attempts to clarify the efficacy of the available over - the - counter options for women seeking self treatment for vaginitis symptoms and further discern which products are appropriate for treating self - diagnosed yeast vaginitis . we visited four common venues frequented by women in their search for vaginitis treatment ; one grocery store , one drug store , one health food store , and the ubiquitous wal - mart . we went to the feminine hygiene section of each store and purchased one of each available product that could be considered a treatment option by the average consumer . cures most vaginal yeast infections were purchased as well as any products claiming to relieve vaginal itching . we next conducted a review of the literature , searching for rcts evaluating the efficacy of each product . our search for published literature included conventional search engines ( pub med , ovid , cochcrane , cinahl , apc journal club , google ) , and alternative medicine databases ( longwood herbal taskforce , nccam , natural medicines com - prehensive database ) . the products purchased could be grouped into three categories : intravaginal imidazoles , vaginal anti - itch creams , and homeopathic treatments . the intravaginal imidazoles were as a whole the more expensive products ranging from $ 6$19 , with an average cost of $ 12 . the homeopathic remedies ranged in price from $ 4$13 , with an average cost of $ 8.50 . finally , the vaginal anti - itch creams range in price from $ 2$6 , with an average price of $ 4 . further , a review of five rcts found no significant difference in treatment durations of one to fourteen days of imidazole therapy for uncomplicated vulvovaginitis . the cdc recommends that pregnant women use the seven - day treatment course due to the lower average concentration of medication . finally , the one , three , and seven - day regimens all deliver the same total dosage of active ingredient ; they differ solely in the number of doses and therefore strength of each dose . our review of the literature failed to reveal studies confirming the efficacy of vaginal anti - itch creams for treatment of infectious vaginitis . these products may confuse women seeking self treatment for vaginitis due to their proximity on the shelves , as well as their claims to relieve vaginal itching , the primary symptom of yeast vaginitis . we could not locate any rcts proving the effectiveness of any of the homeopathic treatments . systemic reviews have not found homeopathy to be a definitively proven treatment for any medical condition . despite the introduction of otc drugs for treatment of vulvovaginitis , the costs of health care office visits to treat this disorder are still rising , to an estimated of 3.1 billion dollars by 2014 . this may be due to the fact that women have proven to be inadequate in self diagnosis . in fact , in one study only one third of the women correctly diagnosed themselves with a yeast vaginitis . further , women with a previous clinical diagnosis of candida infection were not more accurate at identifying their current condition . the most common cause of infectious vulvovaginitis is bacterial vaginosis which has been found to be twice as prevalent as yeast vaginitis . while vaginitis was once thought not to be clinically perilous , there is a mounting body of evidence linking infectious vaginitis with more serious adverse reproductive outcomes . infection with bacterial vaginosis and trichomoniasis have been shown to increase one 's risk of acquiring hiv and other sti 's including pid , which can lead to infertility [ 2 , 9 ] . further , there has been a positive link between bacterial vaginosis and increased risk of preterm labor . bv also increases a women 's risk of postabortion uterine infection and posthysterectomy cuff infection . empowering women to correctly identify and treat their symptoms with the proper medications will alleviate their discomfort and prevent adverse outcomes from lack of recognition of symptoms necessitating a physi- cian visit . since our findings did not prove any significant differences between brand or formulation of any of the intravaginal imidazoles , women should chose their treatment based on personal preference . knowing that these products are equally effective allows the consumer to base her decision on price , route of administration , and ease of use . it should be noted that women with chronic or persistent yeast infections are less likely to respond to short courses of therapy and should consult with their doctor about a specific treatment regimen . vaginal anti - itch creams are grouped on the shelves next to the vaginal yeast infection treatments . these products such as vagisil , vagi - gard , summers eve , and equate vaginal cream are marketed to the consumer as anti - itch creams , making them a seemingly enticing treatment option for symptomatic relief of pruritis associated with candida . these creams utilize ingredients including anesthetics ( benzocaine ) , external analgesics ( resorcinol ) , and anti - pruitics ( hydrocortisone ) . women should be aware that if they purchase these creams they may experience only temporary alleviation of symptoms and this may delay appropriate medical treatment . further , these creams may act as an irritant in some women and in fact exacerbate their symptoms . a wide variety of nontraditional therapies have been touted for their potential to combat yeast infections . the most commonly cited alternative therapies in a literature review were yogurt containing live acidophilus , boric acid tablets , garlic , and tea tree oil . however , our search did not uncover any otc products utilizing these ingredients that bore a label claiming to cure most vaginal yeast infections . of the alternative therapies widely available over - the - counter claiming to treat yeast infections , homeopathic remedies predominate . a variety of homeopathic treatments were not only available at the health food stores , but at least one homeopathic product was on the shelves at each venue we visited , including eckerd drug and wal - mart . homeopathic remedies as a genre were significantly less expensive with a mean price of $ 3.50 less than the intravaginal imidazoles . simplistically , the theory of homeopathy is to administer small doses of toxic substances to stimulate the body 's own immune response . homeopathic products are often so diluted that they no longer contain even a single molecule of the active substance . this accounts for the fact that homeopathic substances have little proven beneficial or harmful effects . studies have found that women with recurrent vaginal yeast infections are more likely to experiment with alternative therapies due to the lower relative cost and the difficulty of obtaining a last minute doctors appointment . an estimated 42% of patients with recurrent vaginal yeast infections have resorted to alternative therapies . the use of products not proven efficacious or approved by the cdc may delay treatment of more serious medical conditions or promote more adverse outcomes . self - treating allows women to minimize out - of - pocket costs , and avoid a costly and time consuming visit to their health care provider . however , choosing a treatment for vaginitis can be cumbersome due to the number of available products , causing the consumer to feel overwhelmed and confused . most topical agents for treating vulvovaginal candidiasis are available otc , packaged in the one , three , or seven - day treatments . these products are of equal efficacy , allowing the consumer to purchase a treatment based on the ease of use and price . consumers should be aware that vaginal anti - itch creams and homeopathic remedies have not - proven efficacy in any rtcs and homeopathic treatments likely do not contain enough active substance to merit an effect . the price discrepancy among treatments may lure the consumer towards treatments that have little or no proven efficacy . it should be noted that these products cost an average of two to three times more than the vaginal anti - itch creams and homeopathic remedies . the authors recommend that physicians be directive in their counseling of patients about which otc products women should purchase once diagnosed with a yeast infection .

the molecular building block approach was employed effectively to construct a series of novel isoreticular , highly porous and stable , aluminum - based metal organic frameworks with soc topology . from this platform , three compounds were experimentally isolated and fully characterized : namely , the parent al - soc - mof-1 and its naphthalene and anthracene analogues . al - soc - mof-1 exhibits outstanding gravimetric methane uptake ( total and working capacity ) . it is shown experimentally , for the first time , that the al - soc - mof platform can address the challenging department of energy dual target of 0.5 g / g ( gravimetric ) and 264 cm3 ( stp)/cm3 ( volumetric ) methane storage . furthermore , al - soc - mof exhibited the highest total gravimetric and volumetric uptake for carbon dioxide and the utmost total and deliverable uptake for oxygen at relatively high pressures among all microporous mofs . in order to correlate the mof pore structure and functionality to the gas storage properties , to better understand the structure property relationship , we performed a molecular simulation study and evaluated the methane storage performance of the al - soc - mof platform using diverse organic linkers . it was found that shortening the parent al - soc - mof-1 linker resulted in a noticeable enhancement in the working volumetric capacity at specific temperatures and pressures with amply conserved gravimetric uptake / working capacity . in contrast , further expansion of the organic linker ( branches and/or core ) led to isostructural al - soc - mofs with enhanced gravimetric uptake but noticeably lower volumetric capacity . the collective experimental and simulation studies indicated that the parent al - soc - mof-1 exhibits the best compromise between the volumetric and gravimetric total and working uptakes under a wide range of pressure and temperature conditions . background . the fda approved over - the - counter ( otc ) use of vaginal antifungals in 1990 . subsequently , a plethora of otc products have become available to women on drugstore shelves . objectives . the purpose of this study was to determine the availability of otc products marketed for the treatment of vaginitis and to determine if their efficacy had been confirmed by published prospective randomized control trials ( rcts ) . materials and methods . the authors chose four retail locations frequented by women seeking vaginitis treatment . all products deemed a viable treatment option were purchased . results . all intravaginal imidazoles purchased , regardless of treatment duration or active ingredient , were found to be of proven efficacy . we were unable to find an rct confirming the effectiveness of vaginal anti - itch creams and homeopathic treatments for vaginitis . conclusion . 45% of products available to women in the feminine hygiene section of the stores surveyed could not be confirmed to be effective for treating infectious vaginitis .

we retrospectively reviewed the cholangiographic findings and hospital records of 67 patients who between january 1997 and may 1999 had undergone metallic biliary stent placement for the palliation of malignant extrahepatic strictures including klatskin tumors and peripheral cholangiocarcinoma invading the confluence of the intrahepatic ducts . patients with recurrence of biliary obstruction after bilioenteric anastomosis ( n = 4 ) or inadequate follow - up for the evaluation of stent patency ( n = 4 ) were excluded , and a total of 59 patients were thus enrolled in this study . thirty - six were male and 23 were female , and their mean age was 63.6 ( range , 25 - 82 ) years . the causes of extrahepatic strictures were as follows : common duct cancer ( n = 5 ) , klatskin tumor ( n = 5 ) , peripheral cholangiocarcinoma invading the hepatic confluence ( n = 5 ) , gall bladder cancer ( n = 5 ) , cancer of the pancreas ( n = 16 ) , and metastasis ( n = 13 ) ( 12 gastric cancer and one adenocarcinoma of unknown origin ) . malignancy was confirmed by surgery in 18 cases , by percutaneous needle biopsy in 19 , by bile cytology with and without brushing in eight , and on the basis of the imaging findings of cholangiography and ct in 14 . in all 59 patients , when reviewing the cholangiograms obtained during percutaneous transhepatic biliary drainage ( ptbd ) and before stent placement , we focused on the angulation formed by the distal and proximal portion of the ehd ( fig . 1 ) and the location of strictures within it ( fig . all cases except one showed a varying degree of angulation ( 80 - 170 ) between the proximal and distal portions of the ehd ; in the exceptional case , the course of the ehd from the biliary confluence to the ampulla of vater was straight . the area of angulation within the ehd was termed ' bending portion ' . to facilitate analysis , the classical method of describing ehd strictures ( 16 ) according to the location of the distal margin of the stricture relative to the ampulla of vater and the bending portion of the ehd , lesions were categorized as one of four types . type a represented a lesion whose distal margin was located within 3 cm of the ampulla of vater , regardless of the bending portion ( fig . 2a ) ; a type - b lesion was one whose distal margin was located more than 3 cm above the ampulla of vater and at or below the bending portion ( fig . 2b ) ; type c represented a lesion whose distal margin was located not more than 3 cm above the bending portion ( fig . 2c ) , while a lesion whose distal margin was located over 3 cm from the bending portion was categorized as type d ( fig . the number of type a , b , c and d cases was 27 , 7 , 9 , and 16 , respectively ( table 1 ) . a case in which a straight extrahepatic duct without angulation was noted was categorized as type a because the stricture was located within 3 cm of the ampulla . fifty - seven patients had undergone ptbd before stent placement , and the remaining two underwent placement as a single step . the mean time lag between ptbd and stent placement was 9.5 ( range , 0 - 25 ) days . informed consent was obtained from all patients . using self - expandable stainless stents ( hanaro stent ; mi tech , seoul , korea ) in 48 patients and nitinol stents ( stentech , seoul , korea ) in 11 , the stainless stent was made of 0.25-mm stainless steel wire bent in a zigzag pattern , with alternating different leg lengths forming a spiral cylindric configuration , and for the nitinol stent , 0.18 or 0.20-mm nitinol wire bent in a mesh was used . the length of stent was determined by the length of the stricture . in order to leave sufficient safety margin at each end of the stent within the bile duct , we generally selected a stent at least 4 cm longer than the stricture . if a stent was placed across the ampulla of vater ( transpapillary placement ) , a longer one , which would cover the distal duct , was chosen . mean stent length was 6.2 ( range , 3 - 8 ) cm for the suprapapillary method , and 7.3 ( range , 5 - 9 ) cm for the transpapillary . the routes of stent insertion were right transhepatic in 25 patients , left transhepatic in 24 , and bilateral trans - hepatic in ten . to simplify analysis , methods were classified only from the viewpoint of location of the distal end of the stent : whether or not it crossed the ampulla of vater . the term ' suprapapillary method ' was used to denote a stent placement technique which did not cross the ampulla of vater regardless of the location of the distal end of the stent within the ehd . the term ' transpapillary method ' denoted a placement technique that crossed the ampulla , the distal end of the stent thus being located in the duodenal lumen . in this way the stent was placed using the suprapapillary method in 35 cases and the transpapillary method in 24 ( table 1 ) . to permit bile drainage until the following morning , an 8.5-fr drainage catheter ( simp - loc ; cook , bloomington , ind . ) was positioned after stent placement , and the tube was then clamped for one day . when no problem was evident after clamping the drainage tube and the cholangiogram indicated good passage of contrast media into the duodenum , the drainage tube was removed . when the remaining waist of the stent hindered the passage of contrast media , as occurred in a limited number of cases , a balloon catheter ( olbert ; boston scientific , watertown , mass , u.s.a . ) with a diameter of 8 or 10 mm was used to relieve the waist . if it was found that a previously inserted stent inadequately decompressed the biliary system , a further stent was placed . many type - a lesions were managed using the transpapillary method , whereas for all type - d lesions , the suprapapillary method was used ( table 1 ) . in the 35 cases in which the superapapillary method was employed initially , another stent was used by way of secondary intervention in five lesions because the initial stent failed to maintain patency . there were four such type - a lesions and one type c. their numbers are shown in parentheses in the table . if passage of contrast media through the stent was poor , the procedure was regarded as inadequate . using the fisher exact test , initial patency rates for both the suprapapillary and transpapillary method were compared for each of the four types . the causes of inadequate procedures were analyzed using the cholangiograms obtained before , during , and after stent placement . regardless of the cause of reobstruction , recurrence of symptoms within two months of initial stent placement was defined as ' early recurrence ' . using fisher 's exact test , early recurrence rates were also compared for each of the four types . the duration of stent patency was defined as the interval between initial placement and recurrence of symptoms because of obstruction . patient survival and duration of stent patency were calculated with regard to type of lesion and the method of stent placement . using the kaplan - meier method , differences in long - term patency were compared . we retrospectively reviewed the cholangiographic findings and hospital records of 67 patients who between january 1997 and may 1999 had undergone metallic biliary stent placement for the palliation of malignant extrahepatic strictures including klatskin tumors and peripheral cholangiocarcinoma invading the confluence of the intrahepatic ducts . patients with recurrence of biliary obstruction after bilioenteric anastomosis ( n = 4 ) or inadequate follow - up for the evaluation of stent patency ( n = 4 ) were excluded , and a total of 59 patients were thus enrolled in this study . thirty - six were male and 23 were female , and their mean age was 63.6 ( range , 25 - 82 ) years . the causes of extrahepatic strictures were as follows : common duct cancer ( n = 5 ) , klatskin tumor ( n = 5 ) , peripheral cholangiocarcinoma invading the hepatic confluence ( n = 5 ) , gall bladder cancer ( n = 5 ) , cancer of the pancreas ( n = 16 ) , and metastasis ( n = 13 ) ( 12 gastric cancer and one adenocarcinoma of unknown origin ) . malignancy was confirmed by surgery in 18 cases , by percutaneous needle biopsy in 19 , by bile cytology with and without brushing in eight , and on the basis of the imaging findings of cholangiography and ct in 14 . in all 59 patients , when reviewing the cholangiograms obtained during percutaneous transhepatic biliary drainage ( ptbd ) and before stent placement , we focused on the angulation formed by the distal and proximal portion of the ehd ( fig . 1 ) and the location of strictures within it ( fig . all cases except one showed a varying degree of angulation ( 80 - 170 ) between the proximal and distal portions of the ehd ; in the exceptional case , the course of the ehd from the biliary confluence to the ampulla of vater was straight . the area of angulation within the ehd was termed ' bending portion ' . to facilitate analysis , the classical method of describing ehd strictures ( 16 ) according to the location of the distal margin of the stricture relative to the ampulla of vater and the bending portion of the ehd , lesions were categorized as one of four types . type a represented a lesion whose distal margin was located within 3 cm of the ampulla of vater , regardless of the bending portion ( fig . 2a ) ; a type - b lesion was one whose distal margin was located more than 3 cm above the ampulla of vater and at or below the bending portion ( fig . 2b ) ; type c represented a lesion whose distal margin was located not more than 3 cm above the bending portion ( fig . 2c ) , while a lesion whose distal margin was located over 3 cm from the bending portion was categorized as type d ( fig . the number of type a , b , c and d cases was 27 , 7 , 9 , and 16 , respectively ( table 1 ) . a case in which a straight extrahepatic duct without angulation was noted was categorized as type a because the stricture was located within 3 cm of the ampulla . fifty - seven patients had undergone ptbd before stent placement , and the remaining two underwent placement as a single step . the mean time lag between ptbd and stent placement was 9.5 ( range , 0 - 25 ) days . informed consent was obtained from all patients . using self - expandable stainless stents ( hanaro stent ; mi tech , seoul , korea ) in 48 patients and nitinol stents ( stentech , seoul , korea ) in 11 , the stainless stent was made of 0.25-mm stainless steel wire bent in a zigzag pattern , with alternating different leg lengths forming a spiral cylindric configuration , and for the nitinol stent , 0.18 or 0.20-mm nitinol wire bent in a mesh was used . the length of stent was determined by the length of the stricture . in order to leave sufficient safety margin at each end of the stent within the bile duct , we generally selected a stent at least 4 cm longer than the stricture . if a stent was placed across the ampulla of vater ( transpapillary placement ) , a longer one , which would cover the distal duct , was chosen . mean stent length was 6.2 ( range , 3 - 8 ) cm for the suprapapillary method , and 7.3 ( range , 5 - 9 ) cm for the transpapillary . the routes of stent insertion were right transhepatic in 25 patients , left transhepatic in 24 , and bilateral trans - hepatic in ten . to simplify analysis , methods were classified only from the viewpoint of location of the distal end of the stent : whether or not it crossed the ampulla of vater . the term ' suprapapillary method ' was used to denote a stent placement technique which did not cross the ampulla of vater regardless of the location of the distal end of the stent within the ehd . the term ' transpapillary method ' denoted a placement technique that crossed the ampulla , the distal end of the stent thus being located in the duodenal lumen . in this way the stent was placed using the suprapapillary method in 35 cases and the transpapillary method in 24 ( table 1 ) . to permit bile drainage until the following morning , an 8.5-fr drainage catheter ( simp - loc ; cook , bloomington , ind . ) was positioned after stent placement , and the tube was then clamped for one day . when no problem was evident after clamping the drainage tube and the cholangiogram indicated good passage of contrast media into the duodenum , the drainage tube was removed . when the remaining waist of the stent hindered the passage of contrast media , as occurred in a limited number of cases , a balloon catheter ( olbert ; boston scientific , watertown , mass , u.s.a . ) with a diameter of 8 or 10 mm was used to relieve the waist . if it was found that a previously inserted stent inadequately decompressed the biliary system , a further stent was placed . many type - a lesions were managed using the transpapillary method , whereas for all type - d lesions , the suprapapillary method was used ( table 1 ) . in the 35 cases in which the superapapillary method was employed initially , another stent was used by way of secondary intervention in five lesions because the initial stent failed to maintain patency . there were four such type - a lesions and one type c. their numbers are shown in parentheses in the table . if passage of contrast media through the stent was poor , the procedure was regarded as inadequate . using the fisher exact test , initial patency rates for both the suprapapillary and transpapillary method were compared for each of the four types . the causes of inadequate procedures were analyzed using the cholangiograms obtained before , during , and after stent placement . regardless of the cause of reobstruction , recurrence of symptoms within two months of initial stent placement was defined as ' early recurrence ' . using fisher 's exact test , early recurrence rates were also compared for each of the four types . the duration of stent patency was defined as the interval between initial placement and recurrence of symptoms because of obstruction . patient survival and duration of stent patency were calculated with regard to type of lesion and the method of stent placement . using the kaplan - meier method , all 24 cases involving the transpapillary method and 23 of 35 ( 65.7% ) in which the suprapapillary method was used showed good initial patency . the difference in initial patency rate between the two methods was statistically significant ( p < .001 ) . in type a , only one ( 14.3% ) involving the seven cases of the suprapapillary method showed good patency , whereas all 20 in which the transpapillary method was employed showed good passage through the stent ( fig . the causes of failure of initial patency were unpredicted stricture of the distal extrahepatic duct near the ampulla of vater in three cases ( fig . 4 ) , aggravation of distal angulation after stenting in two , and misplacement of the stent resulting in inadequate coverage of the stricture at the distal portion of the stent in one . with regard to type - b strictures , four of the five cases involving the suprapapillary method ( 80.0% ) showed good patency ( fig . 5 ) ; the cause of failure of initial patency in one case was aggravation of angulation after stent placement . in two cases the transpapillary method was used , and both were also patent . for type c , three of the seven cases employing the suprapapillary method showed good patency . in the other four , the cause of failure of initial patency was aggravation of the preexisting angulation of the duct due to longitudinal rigidity of the distal portion of the stent ( fig . two cases involved the transpapillary method , and both were also patent . for type d , stricture management relied in all cases on the suprapapillary method , and 15 of 16 ( 93.8% ) showed good patency . the cause of failure of initial patency in one case was aggravation of angulation , which can be prevented if the stent length chosen is appropriate . because of the small sample size , statistical significance for types b , c , and d of the six type - a cases which showed poor patency after initial stent placement , four underwent additional placement across the ampulla of vater , and in two balloon dilatation of the unpredicted stricture of the distal ehd near the ampulla of vater was performed ( fig . 4b ) . in one type - b case of poor patency due to aggravation of angulation at the distal portion of the stent , further intervention was not attempted . among the four type - c cases with poor patency , one underwent transpapillary placement of an additional stent immediately after initial placement , but in the other three there was no further intervention . in one type - d case of poor patency , after reintervention there were changes in the numbers of cases in which the suprapapillary / transpapillary method was employed . the new totals are shown in parentheses ( see table 1 ) . among the 30 cases in which the suprapapillary method was used , there were seven in which early recurrence occurred , including five of poor patency at initial stent placement but without additional placement . there were just two cases of early recurrence in the remaining patients , all of whom showed good patency of the biliary tree after stent placement . in four of 29 cases involving the transpapillary method ( 13.8% ) , early recurrence the six - month and one - year patency rates were 58.9% and 39.4% , respectively , while the median patency intervals for type a , b , and c were 201 , 317 , and 50 days , respectively . we were unable to calculate the median patency interval for type d. all differences were statistically insignificant ( p = .39 ) . the median patency intervals for cases in which the suprapapillary and transpapillary methods were used were 317 and 253 days , respectively , with no significant statistical difference ( p = .57 ) . all 24 cases involving the transpapillary method and 23 of 35 ( 65.7% ) in which the suprapapillary method was used showed good initial patency . the difference in initial patency rate between the two methods was statistically significant ( p < .001 ) . in type a , only one ( 14.3% ) involving the seven cases of the suprapapillary method showed good patency , whereas all 20 in which the transpapillary method was employed showed good passage through the stent ( fig . the causes of failure of initial patency were unpredicted stricture of the distal extrahepatic duct near the ampulla of vater in three cases ( fig . 4 ) , aggravation of distal angulation after stenting in two , and misplacement of the stent resulting in inadequate coverage of the stricture at the distal portion of the stent in one . with regard to type - b strictures , four of the five cases involving the suprapapillary method ( 80.0% ) showed good patency ( fig . 5 ) ; the cause of failure of initial patency in one case was aggravation of angulation after stent placement . in two cases the transpapillary method was used , and both were also patent . for type c , three of the seven cases employing the suprapapillary method showed good patency . in the other four , the cause of failure of initial patency was aggravation of the preexisting angulation of the duct due to longitudinal rigidity of the distal portion of the stent ( fig . two cases involved the transpapillary method , and both were also patent . for type d , stricture management relied in all cases on the suprapapillary method , and 15 of 16 ( 93.8% ) showed good patency . the cause of failure of initial patency in one case was aggravation of angulation , which can be prevented if the stent length chosen is appropriate . because of the small sample size , statistical significance for types b , c , and d of the six type - a cases which showed poor patency after initial stent placement , four underwent additional placement across the ampulla of vater , and in two balloon dilatation of the unpredicted stricture of the distal ehd near the ampulla of vater was performed ( fig . 4b ) . in one type - b case of poor patency due to aggravation of angulation at the distal portion of the stent , further intervention was not attempted . among the four type - c cases with poor patency , one underwent transpapillary placement of an additional stent immediately after initial placement , but in the other three there was no further intervention . in one type - d case of poor patency , after reintervention there were changes in the numbers of cases in which the suprapapillary / transpapillary method was employed . among the 30 cases in which the suprapapillary method was used , there were seven in which early recurrence occurred , including five of poor patency at initial stent placement but without additional placement . there were just two cases of early recurrence in the remaining patients , all of whom showed good patency of the biliary tree after stent placement . in four of 29 cases involving the transpapillary method ( 13.8% ) , early recurrence the six - month and one - year patency rates were 58.9% and 39.4% , respectively , while the median patency intervals for type a , b , and c were 201 , 317 , and 50 days , respectively . we were unable to calculate the median patency interval for type d. all differences were statistically insignificant ( p = .39 ) . the median patency intervals for cases in which the suprapapillary and transpapillary methods were used were 317 and 253 days , respectively , with no significant statistical difference ( p = .57 ) . for the effective palliation of malignant ehd strictures , it is essential to consider the anatomic characteristics of the ehd . these include the length of the bile duct and the location of cystic duct insertion , as well as the size and anatomic level of the obstructing tumor itself within the ehd ( 4 , 8) . for the prevention of tumor overgrowth it is very important to cover the bile duct for as great a disttance as possible ( over - stenting ) , leaving safety margins below and above the tumorous stricture ( 3 , 8) . when stenting the strictures located adjacent to the ampulla of vater , however , it is sometimes impossible to leave adequate distal safety margins . in our series , when the stent was placed by means of the suprapapillary method in such lesions ( type a ) , we were often faced with poor patency just after stent placement ; this was due to unexpected narrowing distal to the stent or angulation of the bile duct below the stent . though we can not adequately explain the presence of an unexpected stricture , it may have been caused by edematous change or spasm of the ampulla of vater associated with the procedure , by extrinsic compression of metastatic lymphadenopathy , or by infiltration of the tumor . in addition , we emphasize the importance of natural bending ( angulation ) of the ehd for successful palliation of the obstruction . the course of the extrahepatic duct from the confluence of the hepatic duct to the ampulla of vater is , in most cases , not straight . anatomically , the supraduodenal and retroduodenal segments of the extrahepatic duct slant obliquely from the right to the left , and below them the pancreatic segment curves rather sharply to the right to enter the descending duodenum via its posteromedial surface ( 18 ) . the overall course of the extrahepatic duct results in convexity or angulation to the left , the degree of angulation varying according to the patient . by reviewing the cholangiographic findings of 270 patients , liu et al . ( 9 ) measured the degree of angulation , the mean value of which was 140 - 150. a tumor such as pancreatic cancer around the duct sometimes aggravates the degree of angulation ( 9 ) . we often encountered cases showing aggravation by the distal portion of the stent itself of preexisting angulation of the ehd after stent placement . sometimes the angulation , which hindered effective bile flow , progressed on follow - up due to the longitudinal stretching force ( straightening ) of the metallic stent . in such cases another stent was required for distal angulation , and the function of ampulla of vater had to be sacrificed . ( 4 ) reported an interesting case of stent malfunction caused by exaggerated angulation at the proximal portion of a stent placed for the palliation of distal ehd stricture . they pointed out certain disadvantages of angulation , such as ineffective drainage and increased likelihood of damage to the bile duct epithelium by sharp wires ( 4 ) . stoker and lameris ( 10 ) encountered two cases of proximal occlusion caused by straightening of the stent , a phenomenon also observed by huibergtse ( 19 ) , using a wallstent . they found that the straightened proximal tip of a stent embedded in the bile duct wall hindered bile flow , and this facilitated sludge formation . stoker and lameris ( 10 ) suggested that in selected cases , a longer stent or one with a smaller diameter and less expansive force should be used . self - expanding metallic stents will eventually straighten with time , to a certain extent , although the longitudinal flexibility of a wallstent is slightly less than that of the stents we used ( 20 , 21 ) . this implies that if the proximal or distal ends of the stent are located near the bending portion of the bile duct when the rule of simple over - stenting is applied , it is important to overcome the problem of angulation . it is therefore wise to choose a stent which is long enough to successfully traverse the bending portion of the bile duct ( 4 ) . we recommend the transpapillary method for the palliation of type - a or c lesions in which the stricture is close to the ampulla of vater or the bile duct is angulated below the stricture . for type - b or d lesions in which the stricture is far from the ampulla of vater or bile duct angulation occurs apart from the stricture so that the stent can be placed without aggravation of preexisting angulation , a conventional suprapapillary method can easily be applied . if the suggested indication is applied , the number of cases amenable to the transpapillary method increases very considerably . ( 22 ) placed transpapillary stents in 22 of their 65 patients , even though the method was applied in restricted cases . when they were sure that the papilla was not involved , suprapapillary placement was attempted . in our series , where our indication was applied , the transpapillary method was indicated in 38 of 59 patients ( 64% ) . although some authors have suggested that duodenal ulceration and subsequent bleeding or perforation of the duodenum , as well as both reflux of duodenal content into the bile duct ( once believed to be a cause of early stent dysfunction ) and pancreatitis are complications of the transpapillary method of stent placement , the cases they encountered were limited ( 12 - 15 ) . in spite of concerns over these complications , most reports describing the long - term results of metallic stent insertion have demonstrated that their incidence is very low ( 2 , 12 ) . interestingly , most reports of bleeding involved cases in which the prototype wallstent was used ( 15 ) . nowadays , wallstents and other commercially available stents have relatively smooth edges and this problem does not , therefore , exist . ( 2 ) reported that they did not encounter such complications , though stoker and lameris ( 10 ) described two cases of duodenal pressure ulcer among 135 patients who had undergone transpapillary wallstent placement . though we were unable to analyze the complications arising from the methods employed in this retrospective study , the transpapillary method gave rise to no serious complication . there were , however , relatively frequent incidences of epigastric discomfort after placement , and these might be caused by inadvertent dilatation of the ampulla of vater by the stent . within two or three days , however , they subsided spontaneously . ( 22 ) , no radiologic report has compared the efficiency with which the suprapapillary and transpapillary methods of stent placement palliate malignant biliary obstruction . ( 22 ) found that the occlusion rate of a stent was not influenced by its position . a further report on the same subject ( 23 ) dealt with endoscopically - placed plastic stents , concluding that there was no significant difference in patency and complication rates . we observed similar results : there were no significant differences in early recurrence and long - term patency rates between the two methods . in conclusion , a careful review of cholangiograms with regard to stricture location and ehd angulation , as well as proper application of stent placement methods , are mandatory procedures if malignant biliary strictures are to be successfully palliated . for strictures involving distal ehd and associated with angulation of the bile duct below the stricture , they may have similar presentations but possibly different treatment modalities , ie , hysterectomy for adenomyosis versus conservative management or hysterectomy for leiomyomas , or both . however , only limited studies have been performed on the efficacy of conservative treatment of adenomyosis , and these typically do not improve or preserve fertility . . mri provides better tissue contrast and may be superior to other imaging modalities in certain pelvic pathologies , such as congenital uterine anomalies , leiomyoma , adenomyosis , and endometriomas . some reports suggest mri is a reliable preoperative tool available to physicians to assist in differentiating between fibroids and adenomyoma / adenomyosis . in this study , we evaluated the predictive value of the pre - operative mri in differentiating between leiomyoma and adenomyosis compared with pathologic findings . a 5-year retrospective chart review was performed from january 1999 to december 2004 in a university - based hospital among patients who were admitted to undergo hysterectomy , myomectomy , or both . although 1517 charts were reviewed , only 153 patients who underwent a preoperative mri were selected for this study . patients ranged in age from 21 to 69 with a mean age of 41 and a median age of 39 . the presenting symptoms were as follows : 73(48% ) had menorrhagia , 34(22% ) had dysmenorrhea , 31(20% ) had pelvic pain , 40(26% ) had a pelvic mass , 16(10% ) had irregular bleeding , 14(9% ) had infertility , and 2(1.3% ) had postmenopausal bleeding . of these patients , 50 had a hysterectomy and the other 103 had a myomectomy . mri studies were performed in axial , sagittal , and coronal planes with various t1-weighted spin - echo and t2-weighted turbo spin - echo or spin - echo on a 1.5 tesla system ( gyro scan , phillips medical systems , bothell , washington , usa or ge open magnet mri scanners , general electric company , fairfield , connecticut , usa ) in different centers . contrast enhanced gadolinium was used as an additive technique in 50 cases to achieve better tissue contrast in the patients suspicious for leiomyoma . leiomyoma appeared as low - signal intensity in t2-weighted images and intermediate- to high - signal intensity in t1-weighted images . adenomyosis was described as a focal or diffuse widening of junctional zone above 12 mm , uterine enlargement , or both , with focal or diffuse low - signal intensity myometrial area in t2-weighted images . also on contrast enhanced t1-weighted images , ( gadolinium scan ) small hypointense myometrial spots mri interpretation was performed by different observers in different centers including private radiology centers , community and university hospitals . mri studies were performed in axial , sagittal , and coronal planes with various t1-weighted spin - echo and t2-weighted turbo spin - echo or spin - echo on a 1.5 tesla system ( gyro scan , phillips medical systems , bothell , washington , usa or ge open magnet mri scanners , general electric company , fairfield , connecticut , usa ) in different centers . contrast enhanced gadolinium was used as an additive technique in 50 cases to achieve better tissue contrast in the patients suspicious for leiomyoma . leiomyoma appeared as low - signal intensity in t2-weighted images and intermediate- to high - signal intensity in t1-weighted images . adenomyosis was described as a focal or diffuse widening of junctional zone above 12 mm , uterine enlargement , or both , with focal or diffuse low - signal intensity myometrial area in t2-weighted images . also on contrast enhanced t1-weighted images , ( gadolinium scan ) small hypointense myometrial spots were indicative of adenomyosis . mri interpretation was performed by different observers in different centers including private radiology centers , community and university hospitals . the results were used to confirm the mri findings for the presence of leiomyoma and adenomyosis . according to the pathology reports , 120 had only leiomyoma , 23 had leiomyoma with coexisting adenomyosis , and 9 had adenomyosis . one patient in the hysterectomy group had a normal mri and pathology report that revealed no myoma or adenomyosis . fifteen patients had other pelvic pathologies coexisting with leiomyoma or adenomyosis , or coexisting with both . mri studies correctly diagnosed 136 of 144 patients with leiomyoma . in the remaining 8 patients with leiomyoma data analyses were as follows : sensitivity 94% , specificity 33% , positive predictive value 95% , and negative predictive value 27% . for adenomyosis , in 31 patients , mri revealed true positive , false - positive , and false - negative results in 12 , 11 , and 19 patients , respectively . the positive and negative predictive values of mri for adenomyosis were 52% , 85% , respectively , with 38% sensitivity and 91% specificity . as mentioned previously mri revealed correct diagnoses in only 9/23 patients , in 12/23 it revealed only myoma , and in 2/23 it diagnosed only adenomyosis . adenomyosis and leiomyoma are 2 benign uterine conditions with similar clinical presentations and different pathogeneses . adenomyosis or internal endometriosis is a disease of parous women with the peak incidence between 40 to 50 years of age . it is characterized by the presence of endometrial glands and stroma inside the uterine myometrium . according to different histological interpretations of the hysterectomy specimens , frequency of adenomyosis uterine involvement may be focal or diffuse , and the size of the lesion ranges from a gross macroscopic well - defined lesion ( adenomyoma that mimics myoma ) to a microscopic lesion . uterine leiomyoma is a benign smooth muscle tumor with 25% prevalence in women of reproductive age with a predominance among african american women . furthermore , pathology examination of surgical specimens revealed an incidence of leiomyoma as high as 77% . a definitive diagnosis for both categories is possible based on histological examination of surgical specimens , but imaging techniques are increasingly used for preoperative evaluation and diagnosis . transvaginal ultrasound and pelvic mri are increasingly used as a preoperative diagnostic tool for adenomyosis . although both techniques have been reported to have almost the same sensitivity ( 68% versus 70% ) , the specificity is greater for mri ( 86% versus 65% ) . pelvic mri is also used in the diagnosis of leiomyoma when a patient 's obesity or extreme uterine retroversion , or both , hinder pelvic ultrasound performance . pelvic mri is highly specific in the diagnosis of adenomyosis ( 91% ) and highly sensitive for leiomyoma ( 94% ) according to our findings . currently , pelvic ultrasound , a relatively affordable and effective method , is widely used as an office procedure in the diagnosis of leiomyoma . it is more efficacious to limit the use of mri for specific conditions when uterine preservation is the goal of therapy , such as localization of leiomyoma in an infertile patient or when the patient is a candidate for uterine artery embolization . our results are different from the results in previously published reports that indicated a higher sensitivity ( 70% vs. 38% ) of mri for adenomyosis and higher specificity ( 100% vs. 33% ) for leiomyoma . this can possibly be explained because our study was influenced by selection bias . because the study was performed on a diseased population with a previous diagnosis of myoma or adenomyosis , or both , where ultrasound alone was thought by the physician to be inadequate . therefore , there were very few patients in our study with negative findings , leading to a low specificity . however , it was not possible to select another population , because of the retrospective nature of the study . also , mri studies were done in different centers and interpreted by different radiologists , which may produce interobserver bias . like other radiological studies , mri is highly operator dependent , and the results may be influenced by technical and observer interpretation . however , this may be a strength of the study in the sense that in reality there will not always be the same specialist reviewing all the studies . of 153 patients who had mri as a preoperative diagnostic tool , 103 underwent myomectomies , so there may be an inherent bias against identifying false - negative adenomyosis . at the present time , pelvic ultrasound is used as a first line for the diagnosis of uterine pathology . however , it may be helpful for diagnosis of pelvic pathologies that are not well defined with ultrasound studies . we suggest mri be used as a complementary diagnostic tool in patients whose ultrasound examination does not clarify the diagnosis , such as in extreme uterine retroversion or obesity , or both , and in patients whose ultrasound examination is indicative of adenomyosis and there is a desire for conservative management . if the preoperative mri shows no adenomyosis , patients can be counseled of the low likelihood of having adenomyosis ( 15% ) and therefore conservative management can be offered .

objectiveto compare the efficacy of suprapapillary and transpapillary methods of transhepatic biliary metallic stent placement in malignant biliary strictures and to specify the indications of each method applied.materials and methodsstents were placed in 59 patients . strictures were categorized as type a ( within 3 cm of the ampulla , n = 27 ) , type b ( over 3 cm from ampulla , n = 7 ) , type c ( within 3 cm of the bending portion , n = 9 ) , or type d ( over 3 cm above the bending portion , n=16 ) . the stenting method was suprapapillary in 34 cases and transpapillary in 25 . the rates of initial and long - term patency and of early recurrence were compared.resultsinitial patency rates for the suprapapillary and transpapillary methods were 1/7 ( 14.3% ) and 20/20 ( 100% ) respectively for type a ( p < 0.0001 ) , 4/5 ( 80.0% ) and 2/2 for type b , 3/7 ( 42.9% ) and 2/2 for type c , and 15/16 ( 93.8% ) and 0/0 for type d. early recurrence rates were 7/30 ( 23.3% ) using the suprapapillary method and 4/29 ( 13.8% ) using the transpapillary method ( p = 0.51 ) . the long - term patency rate did not differ significantly according to either type ( p = 0.37 ) or method ( p = 0.62).conclusionfor good initial patency , the transpapillary method is recommended for strictures of the distal extrahepatic duct near the ampulla and just above the bending portion . long - term patency is not influenced by the stenting method employed . objective : we evaluated the role of mri as a preoperative diagnostic tool for leiomyoma and adenomyosis.method:this is a retrospective chart review at a university - based hospital . the study included 1517 women who underwent hysterectomy or myomectomy over a 5-year period , and 153 women with a preoperative pelvic mri were included . comparisons were made between the results of the mri and postoperative pathology reports.results:the mri and pathology report were the same for 136 of 144 women with leiomyoma and 12 of 31 women with adenomyosis . the mri had 94% sensitivity and 33% specificity for leiomyoma and 38% sensitivity and 91% specificity for adenomyosis . positive and negative predictive values of mri for leiomyoma were 95% and 27% with 90% accuracy . positive and negative predictive values of mri for adenomyosis were 52% and 85% , respectively , with 80% accuracy.conclusion:mri has a high sensitivity and a low specificity for diagnosing leiomyoma and a high specificity and a low sensitivity for diagnosing adenomyosis . due to the high cost and technical variations , we suggest using mri only as an adjunctive diagnostic tool when ultrasound is not conclusive and differentiation between the 2 pathologies ultimately affects patient management .

the utility of this model stems from the fact that few assumptions are needed to determine hazard ratios based on the coefficients . the stratified cox ( sc ) model is a modification of the cox ph model , which allows for control by stratification of a predictor that does not satisfy the ph assumption . parametric models are used only occasionally in analyzing clinical studies of survival despite offering some advantages over semiparametric models . parametric regression analysis is an attractive alternative to the widely used cox model when hazard functions themselves are of primary interest , or when relative survival times are the primary measure of association . when empirical information is available , parametric models can provide insight into the shape of the baseline hazard and baseline survival . furthermore , difficulty choosing the appropriate family of distributions leads many researchers to prefer the cox model . some parametric models are accelerated failure time ( aft ) models which assume that the relationship between the logarithm of survival time and covariates is linear one approach to address these difficulties is fitting the generalized gamma ( gg ) distribution . this extensive family contains nearly all commonly used distributions including the exponential , weibull , and log normal , making it particularly useful for estimating individual hazard functions as well as both relative hazards and relative survival times . in this analysis , we applied both semiparametric and parametric models under different conditions of the ph and aft assumptions and compared their results . the data in this study describe 12,531 women diagnosed with breast cancer in british columbia during 19901999 , and followed till 2010 . these were coded using binary variables equal to one if the subject received the treatment and zero otherwise . we defined survival time as a period between the diagnosis of disease and death or the end of patient 's follow - up . a binary censoring variable was used to indicate whether a patient died of breast cancer . for choosing the appropriate parametric model this distribution is a three - parameter family with location ( ) , scale ( > 0 ) , and shape ( ) parameters , in which all the three parameters depend on covariates . it should be noted that the conventional aft model holds only when covariate effects are modeled through the beta parameter . if we extend the analysis to covariates having effects through the sigma and/or lambda parameters , these are no longer conventional aft models . the gg family contains nearly all of the most commonly used distributions in survival analysis , including the exponential ( = = 1 ) , weibull ( = 1 ) , and log normal ( = 0 ) . if a general parametric distribution includes other distributions as special cases , the general distribution is called a nesting ( larger ) family of the specific distributions . the gg distribution includes three specific distributions , and thus represents a nesting family of them , allowing us to evaluate the appropriateness of the specific distributions relative to each other . testing the appropriateness of a family of distributions is equivalent to testing whether a subset of parameters in its nesting distribution are equal to specific values , and can be performed using a likelihood ratio test . the log - logistic distribution is a commonly used distribution in survival analysis , which is not nested in the gg family . to compare the selected parameter distribution with the log - logistic distribution , we used simple procedures based on baysian information criterion ( bic schwarz , 1978 and akaike information criterion ( aic ; akaike , 1969 ) , in which r is defined as . in our comparisons , the candidate distribution with the largest r value was considered the best fit . for aft models , we can estimate relative survival time by exponentiating the coefficient of a variable . for other models , we can calculate the relative time , rt ( p ) , using appropriate formulae . the relative times are defined for 0 < p < 1 as the ratio of the corresponding quantile functions , rt ( p ) = t1(p)/t0(p ) . the interpretation of rt ( p ) is that the time required for proportion p of individuals in the exposed or treated population to experience the event of interest is rt(p)-fold the time for the same proportion of events to occur in the reference population . links between quantiles of the gamma and gg facilitats use of software to obtain the percentiles of the gg . cox was the first to propose the model , specifying the hazard function as a function of time and the covariates : h ( t , x , b ) = h0(t ) exp(xb ) . with this parameterization , the hazard ratio is : hr ( t , x1 , x0 ) = exp(b(x1 - x0 ) ) . the cox ph model assumes that the hazard ratio for any two specifications of predictors is constant over time , and schoenfeld residuals can be used to assess the ph assumption . the sc model is a modification of the cox ph model , which allows for control by stratification of a predictor that does not satisfy the ph assumption.the predictor that does not satisfy the ph assumption is being adjusted by stratification , whereas the predictor that satisfies the ph assumption is being adjusted by its inclusion in the model . the hazard ratio value for the effect of variables in each stratum can be estimated . nevertheless , the hazard ratio value for the effect of a stratified variable can not be estimated . furthermore , we applied the likelihood ratio ( lr ) test to check the interaction between stratified variable and variables in each stratum . a standard treatment protocol for breast cancer is determined mainly by the patient 's age , stage of cancer , and tumor sensitivity to certain hormones . breast cancer stage has an important role in choosing the treatment , and a patient 's response to treatment depends on her age , so we divided the dataset according to the age of patient at diagnosis of disease ( age < 50 , age 50 ) and the stage of cancer ( i , ii , iii , iv ) . this produced eight combinations of age and stage with different conditions of ph and aft assumptions , and we could compare the treatment effect on patient survival with parametric and semi - parametric models . the presence of hormone receptors has been proven to have an effect on survival time of patient , and was included in all models . for each combination of age and stage , we only included variables for which more than 10 patients received and did not receive the treatment . the data in this study describe 12,531 women diagnosed with breast cancer in british columbia during 19901999 , and followed till 2010 . these were coded using binary variables equal to one if the subject received the treatment and zero otherwise . we defined survival time as a period between the diagnosis of disease and death or the end of patient 's follow - up . a binary censoring for choosing the appropriate parametric model , we started by fitting the saturated gg distribution . this distribution is a three - parameter family with location ( ) , scale ( > 0 ) , and shape ( ) parameters , in which all the three parameters depend on covariates . it should be noted that the conventional aft model holds only when covariate effects are modeled through the beta parameter . if we extend the analysis to covariates having effects through the sigma and/or lambda parameters , these are no longer conventional aft models . the gg family contains nearly all of the most commonly used distributions in survival analysis , including the exponential ( = = 1 ) , weibull ( = 1 ) , and log normal ( = 0 ) . if a general parametric distribution includes other distributions as special cases , the general distribution is called a nesting ( larger ) family of the specific distributions . the gg distribution includes three specific distributions , and thus represents a nesting family of them , allowing us to evaluate the appropriateness of the specific distributions relative to each other . testing the appropriateness of a family of distributions is equivalent to testing whether a subset of parameters in its nesting distribution are equal to specific values , and can be performed using a likelihood ratio test . the log - logistic distribution is a commonly used distribution in survival analysis , which is not nested in the gg family . to compare the selected parameter distribution with the log - logistic distribution , we used simple procedures based on baysian information criterion ( bic schwarz , 1978 and akaike information criterion ( aic ; akaike , 1969 ) , in which r is defined as . in our comparisons , the candidate distribution with the largest r value was considered the best fit . for aft models , we can estimate relative survival time by exponentiating the coefficient of a variable . for other models , we can calculate the relative time , rt ( p ) , using appropriate formulae . the relative times are defined for 0 < p < 1 as the ratio of the corresponding quantile functions , rt ( p ) = t1(p)/t0(p ) . the interpretation of rt ( p ) is that the time required for proportion p of individuals in the exposed or treated population to experience the event of interest is rt(p)-fold the time for the same proportion of events to occur in the reference population . links between quantiles of the gamma and gg facilitats use of software to obtain the percentiles of the gg . cox was the first to propose the model , specifying the hazard function as a function of time and the covariates : h ( t , x , b ) = h0(t ) exp(xb ) . with this parameterization , the hazard ratio is : hr ( t , x1 , x0 ) = exp(b(x1 - x0 ) ) . the cox ph model assumes that the hazard ratio for any two specifications of predictors is constant over time , and schoenfeld residuals can be used to assess the ph assumption . the sc model is a modification of the cox ph model , which allows for control by stratification of a predictor that does not satisfy the ph assumption.the predictor that does not satisfy the ph assumption is being adjusted by stratification , whereas the predictor that satisfies the ph assumption is being adjusted by its inclusion in the model . the hazard ratio value for the effect of variables in each stratum can be estimated . nevertheless , the hazard ratio value for the effect of a stratified variable can not be estimated . furthermore , we applied the likelihood ratio ( lr ) test to check the interaction between stratified variable and variables in each stratum . a standard treatment protocol for breast cancer is determined mainly by the patient 's age , stage of cancer , and tumor sensitivity to certain hormones . breast cancer stage has an important role in choosing the treatment , and a patient 's response to treatment depends on her age , so we divided the dataset according to the age of patient at diagnosis of disease ( age < 50 , age 50 ) and the stage of cancer ( i , ii , iii , iv ) . this produced eight combinations of age and stage with different conditions of ph and aft assumptions , and we could compare the treatment effect on patient survival with parametric and semi - parametric models . the presence of hormone receptors has been proven to have an effect on survival time of patient , and was included in all models . for each combination of age and stage , we only included variables for which more than 10 patients received and did not receive the treatment . for choosing the appropriate parametric model , we started by fitting the saturated gg distribution . this distribution is a three - parameter family with location ( ) , scale ( > 0 ) , and shape ( ) parameters , in which all the three parameters depend on covariates . it should be noted that the conventional aft model holds only when covariate effects are modeled through the beta parameter . if we extend the analysis to covariates having effects through the sigma and/or lambda parameters , these are no longer conventional aft models . the gg family contains nearly all of the most commonly used distributions in survival analysis , including the exponential ( = = 1 ) , weibull ( = 1 ) , and log normal ( = 0 ) . if a general parametric distribution includes other distributions as special cases , the general distribution is called a nesting ( larger ) family of the specific distributions . the gg distribution includes three specific distributions , and thus represents a nesting family of them , allowing us to evaluate the appropriateness of the specific distributions relative to each other . testing the appropriateness of a family of distributions is equivalent to testing whether a subset of parameters in its nesting distribution are equal to specific values , and can be performed using a likelihood ratio test . the log - logistic distribution is a commonly used distribution in survival analysis , which is not nested in the gg family . to compare the selected parameter distribution with the log - logistic distribution , we used simple procedures based on baysian information criterion ( bic schwarz , 1978 and akaike information criterion ( aic ; akaike , 1969 ) , in which r is defined as . in our comparisons , the candidate distribution with the largest r value was considered the best fit . for aft models , we can estimate relative survival time by exponentiating the coefficient of a variable . for other models , we can calculate the relative time , rt ( p ) , using appropriate formulae . the relative times are defined for 0 < p < 1 as the ratio of the corresponding quantile functions , rt ( p ) = t1(p)/t0(p ) . the interpretation of rt ( p ) is that the time required for proportion p of individuals in the exposed or treated population to experience the event of interest is rt(p)-fold the time for the same proportion of events to occur in the reference population . links between quantiles of the gamma and gg facilitats use of software to obtain the percentiles of the gg . cox was the first to propose the model , specifying the hazard function as a function of time and the covariates : h ( t , x , b ) = h0(t ) exp(xb ) . with this parameterization , the hazard ratio is : hr ( t , x1 , x0 ) = exp(b(x1 - x0 ) ) . the cox ph model assumes that the hazard ratio for any two specifications of predictors is constant over time , and schoenfeld residuals can be used to assess the ph assumption . the sc model is a modification of the cox ph model , which allows for control by stratification of a predictor that does not satisfy the ph assumption.the predictor that does not satisfy the ph assumption is being adjusted by stratification , whereas the predictor that satisfies the ph assumption is being adjusted by its inclusion in the model . the hazard ratio value for the effect of variables in each stratum can be estimated . nevertheless , the hazard ratio value for the effect of a stratified variable can not be estimated . furthermore , we applied the likelihood ratio ( lr ) test to check the interaction between stratified variable and variables in each stratum . a standard treatment protocol for breast cancer is determined mainly by the patient 's age , stage of cancer , and tumor sensitivity to certain hormones . breast cancer stage has an important role in choosing the treatment , and a patient 's response to treatment depends on her age , so we divided the dataset according to the age of patient at diagnosis of disease ( age < 50 , age 50 ) and the stage of cancer ( i , ii , iii , iv ) . this produced eight combinations of age and stage with different conditions of ph and aft assumptions , and we could compare the treatment effect on patient survival with parametric and semi - parametric models . the presence of hormone receptors has been proven to have an effect on survival time of patient , and was included in all models . for each combination of age and stage , we only included variables for which more than 10 patients received and did not receive the treatment . results of comparison between parametric regression models have been summarized in table 2 . also , the results of fitting parametric model and cox model are shown in tables 3 and 4 respectively . number of patients receiving treatment by stage at diagnosis comparison between parametric regression models in each category of age and stage relative time estimated in parametric models relative hazard estimated in cox model for patients under age 50 years in disease stages i and iv , the ph and aft assumptions were satisfied . in patients under age 50 years with stage i cancer , the best - fitting parametric model used a conventional gg distribution in which chemotherapy ( p < 0.001 ) and erposneg ( p = 0.002 ) were significant . for the cox model , radiotherapy ( p = 0.001 ) , chemotherapy ( p < 0.001 ) , and hormone therapy ( p = 0.01 ) were significant . 50 years with stage iv cancer , a conventional lognormal model was the best candidate in gg family . the aic and bic criteria were the same for conventional lognormal and conventional log - logistic models , but the cox - snell residual plot indicated better fit for the lognormal . radiotherapy ( p = 0.01 ) , hormone therapy ( p = 0.01 ) , and erposneg(p = 0.01 ) were meaningful in lognormal model . for the cox model , radiotherapy ( p = 0.02 ) and erposneg ( p = 0.001 ) were significant and no interactions were significant in any of the models . snell residual plots for the lognormal , log - logistic , and cox ph model are given in figures 13 . cox - snell residual plot for fitted conventional lognormal model for patients under age 50 years with stage iv cancer cox - snell residual plot for fitted conventional log - logistic model for patients under age 50 years with stage iv cancer cox - snell residual plot for proportional cox model for patients under age 50 years with stage iv cancer for patients aged 50 years or more with stage i cancer , the ph assumption was not satisfied , but the aft assumptions held . in parametric models , the conventional gg had the best fit and radiotherapy ( p = 0.014 ) , chemotherapy ( p < 0.001 ) , hormone therapy ( p 0.001 ) were significant . in semiparametric model , the covariates radiotherapy ( p = 0.003 ) , chemotherapy ( p < 0.001 ) , hormone therapy ( p < 0.001 ) were meaningful ; and since the variable erposneg did not hold the ph assumption , it was lost due to stratification and the effects of other parameters were estimated by stratifying the model by this variable . no interactions were significant in each model . for patients aged 50 years or more with stage iv cancer , none of the ph and aft assumptions held . in parametric model , the gg family fitted better than the log - logistic according to aic and bic criteria . in models based on the gg family , surgery ( p = 0.001 ) , radiotherapy ( p = 0.004 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p < 0.001 ) were significant and the interaction of hormone therapy and erposneg ( p = 0.021 ) was meaningful . since the saturated lognormal is not an aft model , the relative survival time was calculated for the 25 , 50 , and 75 quantiles . surgery ( p = 0.001 ) , radiotherapy ( p = 0.03 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p = 0.001 ) were significant . since the ph assumption only held for surgery , the effect of this variable was estimated by stratifying on erposneg . age50 for all patients with stage ii cancer , and patients aged 50 and older with stage iii cancer , neither the ph nor the aft assumptions were satisfied . in semiparametric models , most variables did not meet the ph assumption and their effect could not be estimated ; in parametric models , the saturated gg fitted better than other distributions . for patients under age 50 years in disease stages i and iv , the ph and aft assumptions were satisfied . in patients under age 50 years with stage i cancer , the best - fitting parametric model used a conventional gg distribution in which chemotherapy ( p < 0.001 ) and erposneg ( p = 0.002 ) were significant . for the cox model , radiotherapy ( p = 0.001 ) , chemotherapy ( p < 0.001 ) , and hormone therapy ( p = 0.01 ) were significant . in both models , in patients under age 50 years with stage iv cancer , a conventional lognormal model was the best candidate in gg family . the aic and bic criteria were the same for conventional lognormal and conventional log - logistic models , but the cox - snell residual plot indicated better fit for the lognormal . radiotherapy ( p = 0.01 ) , hormone therapy ( p = 0.01 ) , and erposneg(p = 0.01 ) were meaningful in lognormal model . for the cox model , radiotherapy ( p = 0.02 ) and erposneg ( p = 0.001 ) were significant and no interactions were significant in any of the models . snell residual plots for the lognormal , log - logistic , and cox ph model are given in figures 13 . cox - snell residual plot for fitted conventional lognormal model for patients under age 50 years with stage iv cancer cox - snell residual plot for fitted conventional log - logistic model for patients under age 50 years with stage iv cancer cox - snell residual plot for proportional cox model for patients under age 50 years with stage iv cancer for patients aged 50 years or more with stage i cancer , the ph assumption was not satisfied , but the aft assumptions held . in parametric models , the conventional gg had the best fit and radiotherapy ( p = 0.014 ) , chemotherapy ( p < 0.001 ) , hormone therapy ( p the covariates radiotherapy ( p = 0.003 ) , chemotherapy ( p < 0.001 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p < 0.001 ) were meaningful ; and since the variable erposneg did not hold the ph assumption , it was lost due to stratification and the effects of other parameters were estimated by stratifying the model by this variable . for patients aged 50 years or more with stage iv cancer , none of the ph and aft assumptions held . in parametric model , the gg family fitted better than the log - logistic according to aic and bic criteria . in models based on the gg family , surgery ( p = 0.001 ) , radiotherapy ( p = 0.004 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p < 0.001 ) were significant and the interaction of hormone therapy and erposneg ( p = 0.021 ) was meaningful . since the saturated lognormal is not an aft model , the relative survival time was calculated for the 25 , 50 , and 75 quantiles . surgery ( p = 0.001 ) , radiotherapy ( p = 0.03 ) , hormone therapy ( p < 0.001 ) , and erposneg ( p = 0.001 ) were significant . since the ph assumption only held for surgery , the effect of this variable was estimated by stratifying on erposneg . age50 for all patients with stage ii cancer , and patients aged 50 and older with stage iii cancer , neither the ph nor the aft assumptions were satisfied . in semiparametric models , most variables did not meet the ph assumption and their effect could not be estimated ; in parametric models , the saturated gg fitted better than other distributions . when ph and aft assumptions were satisfied , both the parametric and semiparametric models were appropriate . the models indicate different significant variables , but parametric models have some advantages over semiparametric models in general . with small sample sizes , the ph assumption is satisfied , some studies indicate that parametric ph models are a better approach than the cox model . further , some studies have shown the robustness of parametric aft models to misspecification because of their log - linear form . finally , one advantage of a parametric model compared to a cox model is that the parametric likelihood accommodates right- , left- , or interval - censored data . the cox likelihood , by contrast , handles right - censored data but does not directly accommodate left- or interval - censored data . when the ph assumption is not satisfied but aft assumptions hold , the parametric model can be used as a substitute for the cox model . when neither the ph nor the aft assumptions hold , a member of the gg distribution , the saturated lognormal , can be used to calculate relative survival times in different quantiles . many settings , including breast cancer analysis , where the proportionality assumption does not hold , the lognormal model has been shown to be appropriate.[1214 ] . a meta - analysis has shown that saturated parametric models provide better results than conventional models for comparing treatments . when the ph and aft assumptions do not hold and the saturated gg distribution fits better than other distributions , the gg family contains many different distributions such as the inverse weibull and inverse lognormal . our analysis applied the most commonly used parametric distributions in survival analysis , but we could not determine the best fit . for one category of age and stage in our study , according to aic and bic criteria , the log - logistic distribution gave the same fit as the lognormal model within the gg family . the log - logistic distribution belongs to the generalized f distribution , which includes the gg distribution . when a member of the gg family does not fit satisfactorily , the best distribution can be found through the generalized f distribution family . if the scales of the parameters in cox 's model and the parametric models differ , neither parameter estimates nor their estimated variances are suitable for comparisons . if the cox model is compared to parametric ph models , the efficiency of parameter estimates can be compared by wald - type statistics . for patients with stage iv cancer , semiparametric and parametric models showed lower hazards and longer survival times for patients receiving treatments than those not receiving treatments . for patients with stage i cancer in both the under 50-year and over 50-year age groups , semiparametric and parametric models showed higher hazards and smaller survival times for patients receiving treatments , which might reflect other covariates that caused the patients to receive a certain treatment . for example , studies have shown the ethnicity of patients affect their use of treatments that are more common in lower stages of cancer . in both parametric and semiparametric models applied to different combinations of age and cancer stage , the expression of hormone receptors was associated with a longer survival time and lower hazard , which has been confirmed in many other studies . a major strength of this study is that we fitted models and performed comparisons using a large set of real - life data from a population - based cancer registry . the major limitation is that our findings describe associations with survival , and not causes of survival . a patient 's survival does not necessarily depend on the treatment they receive ; rather , the treatment that a patient receives often depends on disease characteristics that predict survival . in particular , it would be of interest to determine whether the findings are sustained in more - recent patient cohorts where newer treatments have been used . however , there are some recent studies that describe the factors associated with survival time of patients in developing countries . when ph and aft assumptions were satisfied , semiparametric and parametric models provided two different valid approaches for exploring breast cancer patients survival , and the models can be seen as complementary . when ph assumptions were not satisfied but aft conditions held , the parametric model should be used instead of the cox model . when neither the ph nor the aft assumptions were met , the log normal distribution , a member of the gg family , provided an alternative approach to semiparametric model . more generally , when ph assumptions are not satisfied , parametric models should be considered , whether or not aft assumptions are met . despite many promising lupus treatments reaching early - phase human studies over the past several decades , none except belimumab has yet demonstrated efficacy in phase iii trials . attempts to explain the disappointing results of most clinical trials in lupus have pointed to pitfalls in the application and interpretation of clinical endpoints.1 according to recommendations by the food and drug administration , european league against rheumatism and outcome measures in rheumatology , the ideal endpoint should be able to detect both improvement and worsening in different manifestations and discern acute lupus disease activity from chronic damage and changes related to other causes.2 detection of both improvement and worsening is not possible using a global measure of disease activity such as the systemic lupus erythematosus disease activity index ( sledai ) , and it is not a trivial undertaking even with an organ - based assessment such as the british isles lupus assessment group ( bilag ) since different events within one organ may not be differentiated even with that complex instrument . this led to proposals of composite endpoints to detect improvement without worsening , two of which have already been widely incorporated in clinical trials.35 the sle responder index ( sri ) was derived in part by posthoc analysis of data from a failed phase ii study of belimumab.2 this was subsequently used as the primary outcome measure in two successful phase iii trials ( bliss-52 and bliss-76 ) , leading to belimumab approval by regulatory agencies.3 4 the sri consists of scores derived from the sledai and the bilag index : ( 1 ) 4-point reduction in sledai global score , ( 2 ) no new severe disease activity ( bilag a organ score ) or more than one new moderate organ score ( bilag b ) and ( 3 ) no deterioration from baseline in the physician 's global assessment ( 10% of scale ) . although a requirement for no change in treatment was not formally included in the sri definition , initiation of off - protocol medications defined non - response in the bliss studies . based on the success of the bliss programme , sri is currently being used in a number of clinical trials for sle . nonetheless , the treatment effect achieved by adding belimumab to standard of care using the original sri ( four - point drop in sledai ) was at best modest , and it remains unclear whether this is the optimal discriminatory endpoint . the bilag - based composite lupus assessment ( bicla ) was derived by expert consensus6 and employed as the primary endpoint in the emblem trial of epratuzumab in lupus , where it appeared to discriminate well between standard of care and epratuzumab added to standard of care , at least in some doses tested.5 bicla response is defined as ( 1 ) at least one gradation of improvement in baseline bilag scores in all body systems with moderate or severe disease activity at entry ( eg , all a ( severe disease ) scores falling to b ( moderate ) , c ( mild ) , or d ( no activity ) and all b scores falling to c or d ) ; ( 2 ) no new bilag a or more than one new bilag b scores ; ( 3 ) no worsening of total sledai score from baseline ; ( 4 ) 10% deterioration in physicians global assessment and ( 5 ) no initiation of non - protocol treatment . it should be noted that the emblem protocol was more restrictive than the bliss protocols in the increases allowed in background medications at baseline . the clinical components could be studied in isolation of the medication restrictions in order to simply determine presence or absence of clinical improvement . adding medication restrictions ( as has been done in clinical trials ) response to an intervention given at baseline without clouding that assessment by the impact of other medications that might have been added when patients were not responding to that treatment . direct comparison of bicla and sri has been addressed by only few studies to date,7 8 and the impact of the requirement for no medication changes on the simple determination of improvement has not yet been examined for either instrument . in this real - world exercise , the bicla and sri definitions were compared with physician 's clinical assessment of change in disease state with and without medication restriction rules to distinguish between improvement and response . all patients underwent informed consent procedures and health insurance portability and accountability act disclosures in compliance with good clinical practice . individuals were identified who met the 1997 modified american college of rheumatology classification criteria for sle,9 had two cohort study encounters at which the bilag , sledai and physician 's global assessment ( pga ) had been scored and had a sledai6 at the earlier ( baseline ) visit . in a minor deviation from either the selena - sledai ( used in the sri ) or the sledai-2k ( used in the bicla ) , a hybrid sledai is used for this cohort ( identical to the selena - sledai except for the scoring of proteinuria , which uses the sledai-2k definition ) . during a quality check of the data , where discrepancies were found between sledai and bilag scores and the original clinic note , these were corrected based on the original source documentation . the following data were determined retrospectively based on the medical record : some pga values that had not been scored at the time of the clinic encounters , and an overall physician impression of clinical change at the later encounter ( follow - up visit ) as compared with the first visit , determined by review of clinic notes and laboratory values but without consideration of what treatments had been given in the interim . this was categorised as clinically significant improvement ( physician - rated improvement ( pri ) ) , deterioration or no change . medication changes between baseline and follow - up and at the follow - up visit were recorded , and the sri-4 ( sri ) and bicla composite responses were calculated , both with and without consideration of the medication restrictions that characterised the major clinical trials . sri-3 and sri-5 were computed similarly to sri-4 , except for a minimal three - point or five - point improvement in sledai being required , respectively.2 when medication criteria were applied , improvement criteria were not considered to be met at any follow - up visit where lupus medication changes were made after the initial changes that were instituted at baseline with the exception of nsaids or topical agents . the performance of bicla and sri was compared against clinician - rated improvement . potential causes of discrepancies in each instrument were explored , including the role of each component endpoint . descriptive statistics ( mean sd ) were used to describe measures of disease activity ( pga , sledai , global bilag and number of bilag domains involved ) at baseline and follow - up . comparison of disease activity measures between baseline and follow - up was performed by paired t test . disease activity measures between pri responders that did or did not meet the bicla or sri endpoints were compared by mann spearman 's rank test was used to correlate pri with sri and bicla responses . the number of bilag domains with persistent and/or b scores at follow - up among pri responders stratified by the number of bilag a and/or b scores at baseline was compared by fisher 's exact test . descriptive statistics ( mean sd ) were used to describe measures of disease activity ( pga , sledai , global bilag and number of bilag domains involved ) at baseline and follow - up . comparison of disease activity measures between baseline and follow - up was performed by paired t test . disease activity measures between pri responders that did or did not meet the bicla or sri endpoints were compared by mann spearman 's rank test was used to correlate pri with sri and bicla responses . the number of bilag domains with persistent and/or b scores at follow - up among pri responders stratified by the number of bilag a and/or b scores at baseline was compared by fisher 's exact test . in total , 91 patients eligible for the analysis were identified , including 86 women and 5 men with sle . the mean age at the time of the baseline visit was 41 years ( sd 11.8 ) , ranging from 21 to 68 . also , 46 subjects were caucasian , 22 african - american , 14 native american , 5 caucasian / hispanic and 4 asian . the patients in this analysis were all assessed between june 2009 and april 2012 with a mean interval of 6.24 months between baseline and follow - up visits ( range 125 months ) . in total , 68 patients improved by pri , 17 remained the same and 6 deteriorated . disease activity in patients who did or did not improve by pri was compared using the pga , sledai , global bilag scores and the number of bilag organ domains involved ( table 1 ) . a statistically significant improvement between baseline and follow - up in all disease activity instruments was found in patients improving by pri , but no differences in those indices were observed in group not deemed by the physician to be improved . this supports previous literature that outcome measures discriminate clinically relevant differences.10 11 disease activity at baseline and follow - up among patients clinically improving ( n=68 ) or not ( n=23 ) bilag , british isles lupus assessment group ; pga , physician 's global assessment ; pri , physician - rated improvement ; sledai , systemic lupus erythematosus disease activity index . of 68 patients determined to be improving by the physician ( pri ) , 58 met the clinical criteria for sri and 52 met the bicla endpoint , with sensitivity of agreement with pri 85% and 76% , respectively ( table 2 ) . this evaluation was for improvement not response and did not include the restriction that change of medications over - rules response to the baseline treatment . of the 23 patients not improving by pri , 17 did not meet the sri clinical criteria and 18 did not improve by the bicla ; therefore , specificity of sri and bicla for pri was 74% and 78% . spearman 's rank correlations to pri were sri-3 0.605 , sri-4 0.563 , sri-5 0.541 and bicla 0.492 ( all p values<0.000001 ) . sensitivity and specificity of sri and bicla compared with pri bilag , british isles lupus assessment group ; bilca , bilag - based composite lupus assessment ; non - r , non - responders ; pri , physician - rated improvement ; r , responders ; sri , systemic lupus responder index . of the 10 out of 68 patients improving by pri but not by sri , 7 did meet the bicla improving criteria . these seven sri - negative / bicla - positive discordant cases failed the sri because of less than four - point improvement on sledai , which scores mild , moderate or severe arthritis four points and mild , moderate or severe rash two points ( six individuals had a two - point improvement and one had a three - point improvement ; therefore , all did have complete resolution of at least one feature , but the scores given to these particular features were not high enough to define improvement by sri ) . among 16 patients who improved by pri but these 13 sri - positive / bicla - negative discordant cases failed bicla because of one or more organ systems without even partial improvement , despite complete resolution of at least one other feature counting four points on the sledai . persistent b ( moderate ) scores in the musculoskeletal and mucocutaneous domains were present in six patients each , and there was one persistent b in each of the following domains : gastrointestinal ( lupoid hepatitis ) , cardiorespiratory ( pleurisy ) and renal ( proteinuria ) . one patient had a persistent a ( severe ) score in the cardiorespiratory domain ( interstitial lung disease ) . six of the twenty - three patients who did not improve by pri met the sri criteria , three of whom did not improve by bicla . these three sri / bicla discordant cases achieved four - point reduction in sledai due to resolving mild manifestations ( rash , alopecia , mucosal ulcers ) despite other ongoing ( but not worsening ) more severe organ involvement . five pri failures met the bicla , two of which did not meet the sri . one of these two patients developed new bilag b arthritis , and one new b score is allowed in the bicla definition of improvement . another had only moderate improvement in arthritis ( musculoskeletal bilag a score ( severe arthritis ) decreased to a high b ( significant moderate arthritis ) ) , but the patients were globally judged as overall clinically unchanged . since patients improving by pri often failed bicla ( false negative ) because improvement did not occur in all domains , we hypothesised this may be more common if there is a greater number of organs active at baseline . therefore , the total number of bilag domains involved in bicla improvement versus non - improvement was assessed in those patients meeting pri ( table 3 ) . comparison of disease activity indices between pri responders that met ( n=52 ) or not ( n=16 ) the bicla endpoint * mann whitney rank - sum test . bilag , british isles lupus assessment group ; bilca , bilag - based composite lupus assessment ; non - r , non - responders ; pga , physician 's global assessment ; pri , physician - rated improvement ; r , responders ; sledai , systemic lupus erythematosus disease activity index . bicla responders tended to have fewer domains involved at baseline compared with non - responders , an observation that was more pronounced when only organs with baseline a ( severe disease ) and b ( moderate disease ) were counted . this supports the hypothesis that the more bilag organs are significantly involved at baseline , the more likely it is to have persistent disease in one or more organs at follow - up despite significant improvement in some aspects of the disease and an overall clinical impression of improvement . among patients improving by pri , 33.3% of those with two or more bilag a or b scores at baseline had persistent a or b scores at follow - up ( bicla failure ) compared with 10.3% in those with one or less bilag a or b scores at baseline ( fisher 's exact test , p=0.0924 ) . this suggests that the bicla might be less sensitive at detecting improvement when more organs are involved . pri responders meeting the bicla criteria also had less disease activity at follow - up compared with those not meeting the bicla , evident by the pga , sledai and global bilag scores , as well as the number of bilag organs involved ( p<0.02 for all comparisons ) , indicating the possibility that the bicla may provide some additional meaningful discrimination beyond what is captured by pri or be considered a higher bar for determining improvement . disease activity indices at baseline and follow - up were not as informative when comparing those improved by pri who did or did not meet the sri criteria ( table 4 ) . with the exception of pga , there was no difference in baseline disease activity measures in those improving by sri versus failures . comparison of disease activity indices between pri responders that met ( n=58 ) or not ( n=10 ) the sri endpoint * mann whitney rank - sum test . bilag , british isles lupus assessment group ; non - r , non - responders ; pga , physician 's global assessment ; pri , physician - rated improvement ; r , responders ; sledai , systemic lupus erythematosus disease activity index ; sri , systemic lupus responder index . clinical trial endpoints using the sri and bicla constructs to determine response to a treatment initiated at baseline have used restrictions in rescue medications in the definition of response ( underscoring a distinction between the concepts of improvement and response ) . a requirement for no off - protocol medication changes was included in the bicla response definition , used in the embem trial.5 in the bliss trials of belimumab in sle where the sri was used,3 4 medication increases were restricted during the latter months , and protocol deviations defined non - response accomplishing a similar end , although medication restrictions were less restrictive in those protocols than in the emblem study . to examine the impact of medication restrictions on how bicla and sri compare to physician 's determination of improvement , we performed the sri and bicla determination in this real - world sample of patients ( no protocolised restriction on treatment ) . all pharmacological changes in treatment after baseline interventions and prior to and/or at follow - up visit , except for nsaids or topical agents , excluded the designation of improvement by bicla or sri in this analysis . as expected , medication restrictions dramatically decreased the number of patients meeting bicla and sri response definitions . surprisingly , when medications were factored in as response criteria , this increased specificity for pri ( decreased the likelihood that bicla and sri will detect improvement when the physician did not agree ) , even though the physician was not considering the treatment changes in the pri assessment ( table 5 ) . sensitivity and specificity of sri and bicla with medication criteria bilca , british isles lupus assessment group - based composite lupus assessment ; non - r , non - responders ; pri , physician - rated improvement ; r , responders ; sri , systemic lupus responder index . in total , 91 patients eligible for the analysis were identified , including 86 women and 5 men with sle . the mean age at the time of the baseline visit was 41 years ( sd 11.8 ) , ranging from 21 to 68 . also , 46 subjects were caucasian , 22 african - american , 14 native american , 5 caucasian / hispanic and 4 asian . the patients in this analysis were all assessed between june 2009 and april 2012 with a mean interval of 6.24 months between baseline and follow - up visits ( range 125 months ) . in total , 68 patients improved by pri , 17 remained the same and 6 deteriorated . disease activity in patients who did or did not improve by pri was compared using the pga , sledai , global bilag scores and the number of bilag organ domains involved ( table 1 ) . a statistically significant improvement between baseline and follow - up in all disease activity instruments was found in patients improving by pri , but no differences in those indices were observed in group not deemed by the physician to be improved . this supports previous literature that outcome measures discriminate clinically relevant differences.10 11 disease activity at baseline and follow - up among patients clinically improving ( n=68 ) or not ( n=23 ) bilag , british isles lupus assessment group ; pga , physician 's global assessment ; pri , physician - rated improvement ; sledai , systemic lupus erythematosus disease activity index . of 68 patients determined to be improving by the physician ( pri ) , 58 met the clinical criteria for sri and 52 met the bicla endpoint , with sensitivity of agreement with pri 85% and 76% , respectively ( table 2 ) . this evaluation was for improvement not response and did not include the restriction that change of medications over - rules response to the baseline treatment . of the 23 patients not improving by pri , 17 did not meet the sri clinical criteria and 18 did not improve by the bicla ; therefore , specificity of sri and bicla for pri was 74% and 78% . spearman 's rank correlations to pri were sri-3 0.605 , sri-4 0.563 , sri-5 0.541 and bicla 0.492 ( all p values<0.000001 ) . sensitivity and specificity of sri and bicla compared with pri bilag , british isles lupus assessment group ; bilca , bilag - based composite lupus assessment ; non - r , non - responders ; pri , physician - rated improvement ; r , responders ; sri , systemic lupus responder index . of the 10 out of 68 patients improving by pri but not by sri , 7 did meet the bicla improving criteria . these seven sri - negative / bicla - positive discordant cases failed the sri because of less than four - point improvement on sledai , which scores mild , moderate or severe arthritis four points and mild , moderate or severe rash two points ( six individuals had a two - point improvement and one had a three - point improvement ; therefore , all did have complete resolution of at least one feature , but the scores given to these particular features were not high enough to define improvement by sri ) . among 16 patients who improved by pri but not bicla , these 13 sri - positive / bicla - negative discordant cases failed bicla because of one or more organ systems without even partial improvement , despite complete resolution of at least one other feature counting four points on the sledai . persistent b ( moderate ) scores in the musculoskeletal and mucocutaneous domains were present in six patients each , and there was one persistent b in each of the following domains : gastrointestinal ( lupoid hepatitis ) , cardiorespiratory ( pleurisy ) and renal ( proteinuria ) . one patient had a persistent a ( severe ) score in the cardiorespiratory domain ( interstitial lung disease ) . six of the twenty - three patients who did not improve by pri met the sri criteria , three of whom did not improve by bicla . these three sri / bicla discordant cases achieved four - point reduction in sledai due to resolving mild manifestations ( rash , alopecia , mucosal ulcers ) despite other ongoing ( but not worsening ) more severe organ involvement . five pri failures met the bicla , two of which did not meet the sri . one of these two patients developed new bilag b arthritis , and one new b score is allowed in the bicla definition of improvement . another had only moderate improvement in arthritis ( musculoskeletal bilag a score ( severe arthritis ) decreased to a high b ( significant moderate arthritis ) ) , but the patients were globally judged as overall clinically unchanged . since patients improving by pri often failed bicla ( false negative ) because improvement did not occur in all domains , we hypothesised this may be more common if there is a greater number of organs active at baseline . therefore , the total number of bilag domains involved in bicla improvement versus non - improvement was assessed in those patients meeting pri ( table 3 ) . comparison of disease activity indices between pri responders that met ( n=52 ) or not ( n=16 ) the bicla endpoint * mann whitney rank - sum test . bilag , british isles lupus assessment group ; bilca , bilag - based composite lupus assessment ; non - r , non - responders ; pga , physician 's global assessment ; pri , physician - rated improvement ; r , responders ; sledai , systemic lupus erythematosus disease activity index . bicla responders tended to have fewer domains involved at baseline compared with non - responders , an observation that was more pronounced when only organs with baseline a ( severe disease ) and b ( moderate disease ) were counted . this supports the hypothesis that the more bilag organs are significantly involved at baseline , the more likely it is to have persistent disease in one or more organs at follow - up despite significant improvement in some aspects of the disease and an overall clinical impression of improvement . among patients improving by pri , 33.3% of those with two or more bilag a or b scores at baseline had persistent a or b scores at follow - up ( bicla failure ) compared with 10.3% in those with one or less bilag a or b scores at baseline ( fisher 's exact test , p=0.0924 ) . this suggests that the bicla might be less sensitive at detecting improvement when more organs are involved . pri responders meeting the bicla criteria also had less disease activity at follow - up compared with those not meeting the bicla , evident by the pga , sledai and global bilag scores , as well as the number of bilag organs involved ( p<0.02 for all comparisons ) , indicating the possibility that the bicla may provide some additional meaningful discrimination beyond what is captured by pri or be considered a higher bar for determining improvement . disease activity indices at baseline and follow - up were not as informative when comparing those improved by pri who did or did not meet the sri criteria ( table 4 ) . with the exception of pga , there was no difference in baseline disease activity measures in those improving by sri versus failures . comparison of disease activity indices between pri responders that met ( n=58 ) or not ( n=10 ) the sri endpoint * mann whitney rank - sum test . bilag , british isles lupus assessment group ; non - r , non - responders ; pga , physician 's global assessment ; pri , physician - rated improvement ; r , responders ; sledai , systemic lupus erythematosus disease activity index ; sri , systemic lupus responder index . clinical trial endpoints using the sri and bicla constructs to determine response to a treatment initiated at baseline have used restrictions in rescue medications in the definition of response ( underscoring a distinction between the concepts of improvement and response ) . a requirement for no off - protocol medication changes was included in the bicla response definition , used in the embem trial.5 in the bliss trials of belimumab in sle where the sri was used,3 4 medication increases were restricted during the latter months , and protocol deviations defined non - response accomplishing a similar end , although medication restrictions were less restrictive in those protocols than in the emblem study . to examine the impact of medication restrictions on how bicla and sri compare to physician 's determination of improvement , we performed the sri and bicla determination in this real - world sample of patients ( no protocolised restriction on treatment ) . all pharmacological changes in treatment after baseline interventions and prior to and/or at follow - up visit , except for nsaids or topical agents , excluded the designation of improvement by bicla or sri in this analysis . as expected , medication restrictions dramatically decreased the number of patients meeting bicla and sri response definitions . surprisingly , when medications were factored in as response criteria , this increased specificity for pri ( decreased the likelihood that bicla and sri will detect improvement when the physician did not agree ) , even though the physician was not considering the treatment changes in the pri assessment ( table 5 ) . sensitivity and specificity of sri and bicla with medication criteria bilca , british isles lupus assessment group - based composite lupus assessment ; non - r , non - responders ; pri , physician - rated improvement ; r , responders ; sri , systemic lupus responder index . the performance of two composite indices of lupus disease improvement and/or response was compared using as reference a clinician 's global rating of improvement . although the pga , when performed in a paper patient exercise , has been demonstrated to have poor agreement between different physicians,12 a retrospective overall determination of whether or not a patient has a clinically meaningful improvement might be a valuable tool when determined in a consistent manner by one qualified assessor . this was supported in the current exercise , where changes in disease activity ( including sledai and bilag performed prospectively at the time of the visits ) were consistent with the global clinician ratings . retrospectively assessing clinical change between visits is possibly limiting the validity of our observation and warrants conformation prospectively . although the visits used to determine improvement or response were separated by various timepoints in the current study , this did not affect the global clinical assessment of change between visits and is consistent with the manner in which sri and bicla are used in clinical trials as landmark assessments performed at different timepoints compared with a baseline visit . the assessment of response to an intervention is not the same as a measurement of clinical improvement ( which may occur without response to a given intervention if rescue treatments have been given ) . a comparison of bicla and sri without using the medication criteria for response to the baseline treatment was first performed to determine their utility in simply defining clinical improvement . in this assessment , when only those patients ranked as improved by the physician were evaluated , bicla but not sri seemed to define patients with greater improvement in disease activity , suggesting the possibility that it could be more discriminatory in some settings . since more organs involved at baseline decreased the sensitivity of the bicla , it can be hypothesised that this instrument might be particularly useful in patients with less widespread organ involvement , including those patients considered suitable for clinical trials in which less background medication is allowed . in fact , in the emblem trial of epratuzumab in lupus , which limited background medication changes more restrictively than the belimumab trials , the bicla response ( including the medication criteria ) at 12 weeks effectively discriminated the 2400 mg / month cumulative epratuzumab dosage groups from placebo with placebo responses lower than in the bliss trials.5 a comparison of two outcome measures used in different trials does not account for potential differences in patient populations , background treatments or efficacy of the test articles . however , it is worth observing that the apparent improved discriminatory capacity in the emblem trial ( using the bicla ) did not appear to be due to increased efficacy rates in the treatment group but to lower rates in the placebo ( standard of care ) group,7 which does not suggest increased sensitivity in detection of improvement , but might either reflect a more generally ill population , less background treatments or , indeed , the possibility of increased specificity of the improvement measurement . in posthoc analysis of this same trial dataset using the sri,7 sri rates were higher than bicla rates in all arms , including the placebo group , losing discriminatory capacity with a loss of significant differences between treatment and placebo . disagreement between bicla and sri in the emblem trial may , however , have been driven by the baseline distribution of items with high sledai weights that tended to improve at follow - up , with the greatest difference in groups with activity scored as eight - point sledai items ( vasculitis , lupus headache ) at baseline . scoring sledai descriptors for mild / moderate cutaneous vasculitis and headache risks sledai scores that are high in relation to the degree of illness of the patients . sri discriminatory performance might improve in a protocol , restricting the scoring of these features . in the current analysis , no lupus headaches were scored ( consistent with the evaluation here that they are very rare ) , and although four individuals had cutaneous vasculitis at baseline , this resolved in only one at follow - up , thereby not accounting for discrepancies in sri and bicla performance found here . some pri responders failed the sri , providing potential insight into limitations of the sri . patients rated as improving by the physician usually fail to meet the sri improvement criteria due to less than four - point improvement in sledai , which requires not only improvement but complete resolution of at least one manifestation . as expected , this limitation was less evident for sri-3 at the expense of lower specificity for the detection of pri , whereas the opposite was the case for the sri-5 . our results are consistent with the posthoc sensitivity analysis of data from the bliss-76 trial , where modifications of the sri using higher thresholds for selena - sledai improvement ( 5-point to 10-point reductions ) increased differentiation of belimumab from placebo at 52 and 76 weeks but occurred less frequently.4 in an assessment using a physician 's global rating of clinically significant improvement , the bicla may be less sensitive than the sri , particularly in determining improvement in patients with sle with multiple organs involved at baseline . on the other hand , the bicla may be more discriminatory than the sri in selecting those patients with a greater change in disease activity . when sri and bicla were discrepant , this was usually due to the bicla requiring only partial improvement but in all organs versus the sri requiring full improvement and not necessarily in all organs . each instrument could likely be an optimal primary endpoint depending on the population under study and the design of a given clinical trial .

background : the goal of this study is to extend the applications of parametric survival models so that they include cases in which accelerated failure time ( aft ) assumption is not satisfied , and examine parametric and semiparametric models under different proportional hazards ( ph ) and aft assumptions.methods:the data for 12,531 women diagnosed with breast cancer in british columbia , canada , during 19901999 were divided into eight groups according to patients ages and stage of disease , and each group was assumed to have different aft and ph assumptions . for parametric models , we fitted the saturated generalized gamma ( gg ) distribution , and compared this with the conventional aft model . using a likelihood ratio statistic , both models were compared to the simpler forms including the weibull and lognormal . for semiparametric models , either cox 's ph model or stratified cox model was fitted according to the ph assumption and tested using schoenfeld residuals . the gg family was compared to the log - logistic model using akaike information criterion ( aic ) and baysian information criterion ( bic).results : when ph and aft assumptions were satisfied , semiparametric and parametric models both provided valid descriptions of breast cancer patient survival . when ph assumption was not satisfied but aft condition held , the parametric models performed better than the stratified cox model . when neither the ph nor the aft assumptions were met , the log normal distribution provided a reasonable fit.conclusions:when both the ph and aft assumptions are satisfied , the parametric and semiparametric models provide complementary information . when ph assumption is not satisfied , the parametric models should be considered , whether the aft assumption is met or not . objectivesto compare two measures of systemic lupus erythematosus ( sle ) response : the british isles lupus assessment group ( bilag)-based composite lupus assessment ( bicla ) and the systemic lupus responder index ( sri ) against a clinician 's assessment of improvement.methodsninety-one lupus patients were identified with two visits at which systemic lupus erythematosus disease activity index ( sledai ) and bilag had been scored and with active disease ( sledai6 ) at the first visit . a physician rated the disease activity at the second visit as clinically significant improvement , no change or worsening . sri and bicla were scored both with and without the medication criteria often used in trials to restrict response definitions.results68 patients were considered improved , 17 same and 6 worse at follow - up . sri versus bicla , performed without considering medication changes , captured physician - rated improvement with 85% vs 76% sensitivity and 74% vs 78% specificity . with medication limits both instruments had 37% sensitivity and 96% specificity for physician - assessed improvement . seven patients considered improved by the clinician met the bicla but not the sri definition of improvement by failing to achieve a four - point improvement in sledai . 13 clinician - rated responders met sri but not bicla by improving in less than all organs.conclusionsshortfalls of sri and bicla may be due to bicla only requiring partial improvement but in all organs versus sri requiring full improvement in some manifestation(s ) and not all organs . sri and bicla with medication restrictions are less likely to denote response when the physician disagrees and could provide stringent proof of efficacy in appropriately powered clinical trials .

treatment of elbow dislocation with irreparable radial head fracture needs replacement of radial head to achieve stability of elbow . an alternate method in cases of elbow dislocation with radial head fracture can be resection of radial head with repair of medial collateral ligament . we report a retrospective analysis of cases of elbow dislocation with irreparable radial head treated by excision head of radius and repair of mcl . nine patients of elbow dislocation with associated irreparable fractures of the head of the radius were included in this analysis ( 6 f:3 m , age : 35 - 47 years ) . radial head excision was done through the lateral approach and mcl was sutured using no 3 ethibond using medial approach . above elbow plaster all patients were assessed at final followup using mayo elbow performance score ( meps ) . there was no extension deficit when compared to opposite side with mean range of flexion of 138.8 6.97 ( range 130 -145 ) . mean pronation was 87.7 4.4 ( range 80 - 90 ) and mean supination was 87.7 4.62 ( range 80 - 90 ) . radial head excision with mcl repair is an acceptable option for treatment of patients with elbow dislocation and irreparable radial head fracture . elbow dislocations without any associated fracture need no special care except closed reduction under sedation or short anesthesia , splintage for a few days followed by active mobilization for elbow rehabilitation.1 however , those with associated fracture head of radius need repair of head radius or replacement of the radial head along with the reduction of the dislocation to achieve a stable reduction . results of repair or replacement of radial head are not always satisfactory for supination pronation movements , although , the elbow remains stable.2 excision of isolated fracture of the head radius without elbow dislocation gives very satisfactory results,3 as mcl is always intact , though proximal migration of radius after many years has been reported.456 while , excision of head of radius alone , in elbow dislocation gives unsatisfactory results as elbow remain unstable due to loss of both stabilizers of the elbow i.e. head of radius , and mcl.7 hence if the radial head is repairable , it must be repaired in such situation.8 elbow dislocation , with irreparable fracture of the head of radius , needs replacement of the radial head to achieve stable elbow . if , decision is done to excise , isolated head of the radius fracture without dislocation , it must be confirmed under anesthesia , that elbow is stable , and it is not a case of spontaneous , reduction of elbow before patient has reported to the health care services . irreparable fracture head of the radius is said to be present when there are more than three fragments.2 it also becomes irreparable in the absence of adequate implants on the operating table as well as with a surgical team lacking experience.9 instead of doing inadequate fixation which gives restriction of rotation , it is safer to excise the head and repair the mcl . in elbow dislocation , the mcl is always injured.1 if excision of radial head is done for irreparable fracture , there is an increased chance of redislocation of elbow . results of replacement of radial head are not consistent and many long term and short - term failures are reported.8 repair of the comminuted fracture is technically difficult.10 the internal fixation should be reserved for minimally comminuted fractures with three or fewer articular fragments , in associated fracture - dislocations of the elbow.2 when the mcl is not injured in an isolated fracture of the head of the radius , head excision gives consistent results.3 similarly in an elbow dislocation , when the head of the radius is irreparable and needs replacement , if the radial head is excised along with repair of the mcl , it mimics the isolated radial head fracture situation and gave consistently good results.2 we report a retrospective analysis of cases of elbow dislocation with irreparable radial head treated by excision head of radius and repair of mcl . nine patients of elbow dislocation with associated irreparable fractures of the head of the radius were included in this analysis ( 6 f;3 m , age : range 35 - 47 years ) . two cases had an associated type grade 1 coronoid fracture which did not need repair . all elbow injuries with simple dislocations without fracture head of the radius , cases where the head was repairable , cases of terrible triad elbow which needs complex treatment and open fractures were excluded from this analysis . all patients presented immediately after injury . the elbow dislocation was confirmed on plain x - rays . elbow was reduced under sedation in all cases except one where short general anesthesia was used . the exact fracture pattern was assessed on post reduction x - rays [ figure 1 ] . x - ray elbow joint lateral ( a , b ) and anteroposterior ( c ) views of a 47 year old male ( case 6 ) presented with acute injury showing right elbow dislocation and displaced fractures radial head . closed reduction was done to relocate the joint and medial laxity can be well appreciated after reduction the joint was exposed using the lateral approach under general anesthesia and tourniquet . mcl was exposed , by a medial approach , remaining anterior to the medial condyle , avoiding the ulnar nerve . it was observed that the mcl was completely torn in all cases [ figures 25 ] . medial flexor muscles were also injured in all cases ; in six of them , it was injured in the muscle bulk , and in three cases , it had avulsed from the medial condyle . x - ray elbow joint anteroposterior and lateral views of a 36 year old female with elbow dislocation showing relocation of elbow with comminuted fracture radial head intraoperative demonstration of gross medial laxity ( arrow is on medial side ) intraoperative photographs excision of the radial head ( a ) showing lateral approach to the lebow with damaged soft tissues ( b ) irreparable excised radial head intraoperative photographs showing ( a ) the two ends of medial collateral ligament that are approximated ( b ) appearance after suturing the medial collateral ligament mcl with thick nonabsorbable no 3 ethibond sutures and medial stability was established ( c ) appearance after suturing the medial colareral ligament the mcl was sutured with thick nonabsorbable 03 number ethibond ( johnson and johnson , new jersey , usa ) sutures and medial stability was established . muscles , which were torn , were sutured . an above - elbow plaster slab was given for 6 weeks , for soft tissue healing . all patients were followed up till 4 months after removal of plaster , and were assessed for range of motion and elbow stability . all the patients were available for followup with mean followup of 19.55 7.12 months ( range 14 - 36 months ) . no patients reported pain at elbow or any subjective instability . there was no extension deficit when compared to opposite side with mean flexion of 138.8 6.97 ( range 130 - 145 ) . mean pronation was 87.7 4.4 ( range 80 - 90 ) and mean supination was 87.7 4.62 ( range 80 - 90 ) [ table 1 ] . the mean meps was 98.8 3.33 ( range 90 - 100 ) [ table 2 ] . no patient had any sensory complaints , or subluxation , or redislocation till final followup . none of the patients had to change their occupation and all were able to carry out their daily activities without disability . the primary stabilizers of the elbow are the intact bones , namely , olecranon and coronoid , head of radius , and lower end of the humerus . the radial head provides 30% of the valgus stability . with an intact mcl complex , removal of head of radius results in no instability . irreparable fracture head radius is said to be present when there are more than three fragments.2 the repair of multi fragment head of radius is demanding procedure as it requires quality implants and surgical skills of high order . this injury used to be treated by keeping the shattered head intact and elbow cast immobilization for 4 weeks when radial head prosthesis was not available . the delayed excision of radial head was after about 2 to 3 months was advocated to regain supination / pronation . unfortunately , range of motion did not return fully in these cases and poor results were more frequent.12 later , time , head of the radius was excised at time of primary treatment , and elbow was stabilized by steinmann pin till soft tissue healing ( 3 weeks ) . however , it was observed that the elbow could not be maintained in a stable position and had restriction of movements . results improved with the excision of the head of the radius and repaire of the mcl . fracture of the head of the radius can not be excised with associated elbow dislocation , where the mcl is always torn . if the head is excised , both primary and secondary stabilizers are lost and the elbow usually re - dislocates even in plaster . so if any one of the stabilizer is intact , the elbow remains stable like in the case of an excised head without dislocation where the mcl is intact . likewise , if the irreparable head of radius is replaced without repairing the torn mcl , the elbow remains stable . however , controversy still exists regarding which fractures are optimally treated with reduction and internal fixation as well as whether a fracture may be too comminuted to fix . tashjian and katarincic7 concluded that the radial head is a constraint secondary to the mcl for both valgus displacement and internal rotation . isolated repair of the ligament was superior to isolated prosthetic replacement and may be sufficient to restore valgus and internal rotatory stability after excision of the radial head in mcl deficient elbows . moro et al.8 opined that the radial head should be preserved , if possible , to lower the risk and prevent severe instability ; and if the radial head must be resected , suturing of torn ligaments and muscles at the epicondyles was essential . ring et al.9 reported that at the 12-year followup , following open reduction and internal fixation of radial head fractures , 80% had good or excellent results ; however , only 30% were completely pain - free , and 45% had evidence of arthritis . to conclude , repairing the mcl is a better , easier , and more predictable approach , when irreparable head radius fractures needs excision , in complex elbow dislocation . ld is a nationally notifiable condition , and cases are reported by state and local health departments to the centers for disease control and prevention ( cdc ) through the national notifiable diseases surveillance system in accordance with previously established protocols ( 7 ) . , a revised case definition was implemented that altered the laboratory criteria and distinguished between confirmed and probable cases ; cases reported during 20082013 included both categories ( 8) . weighting was applied to state- and county - specific numbers of cases to account for variations in completeness of ethnicity data . descriptive statistics and comparisons were calculated by using sas version 9.3 ( sas institute , cary , nc , usa ) . we compared median age of hispanics and non - hispanics with ld using the kolmogorov - smirnov 2-sample test . cdc human subjects review of the protocol determined it was not research involving human subjects . a total of 374,338 ld cases were reported to cdc , of which 148,444 ( 39.7% ) reports contained information about ethnicity and were included in this analysis . among these , most ( 54.8% ) hispanics with ld were male ; median age was 32 years ( interquartile range 1546 years ) . annual incidence of reported ld among hispanics was 0.8 cases/100,000 persons , compared with 4.0/100,000 among non - hispanics . during 20002001 , hispanics comprised 2.8% of all persons with ld , whereas during 20092013 , hispanics comprised 3.7%4.9% of persons with reported ld . in comparison , the proportion of hispanics in the us population increased slightly during this period , from 13% in 2000 to 16% in 2010 ( 5 ) . although a bimodal age distribution was evident among both hispanics and non - hispanics with ld , the peak in children was less pronounced among hispanics ( figure ) . highest incidence among hispanic children was in boys 1014 years of age , whereas among non - hispanic children , incidence was highest in boys 59 years of age . in adults , highest incidence among both hispanics and non - hispanics was in men 6574 years of age . age- and sex - specific incidence of lyme disease among hispanics ( a ) and non - hispanics ( b ) , united states , 20002013 . for persons hispanics were significantly less likely than non - hispanics to have disease onset during the summer months ( rr 0.85 , 95% ci 0.830.88 ) and more likely to have disease onset during the fall months ( rr 1.15 , 95% ci 1.071.24 ) ( table 1 ) . although erythema migrans was the most commonly reported clinical feature for both groups , it was less commonly reported among hispanics than non - hispanics ( rr 0.83 , 95% ci 0.800.86 ) . manifestations of disseminated disease , such as arthritis and facial palsy , were more commonly reported among hispanics than non - hispanics ( table 1 ) . iqr , interquartile range ; ld , lyme disease ; rr , risk ratio . percentage of persons with ld for whom sex is known ( n = 5,442 hispanics , n = 142,625 non - hispanics ) . the substantial difference in median age between the us hispanic population ( 27 y ) and the us non - hispanic population ( 42 y ) most likely accounts for the difference seen here . defined as 1 of the 13 highest - incidence states that accounted for 95% of all reported confirmed cases of ld in 2010 : connecticut , delaware , maine , maryland , massachusetts , minnesota , new hampshire , new jersey , new york , pennsylvania , vermont , virginia , and wisconsin . overall were reported from high - incidence states , although hispanics with ld were slightly less likely to report residence in a high - incidence state ( rr 0.90 , 95% ci 0.820.98 ) . all of the statistical associations were similar when analysis was restricted to confirmed cases only , with the exception of residence in a high - incidence state , which became nonsignificant ( rr 0.99 , 95% ci 0.891.10 ) . after weighting , nearly half of all estimated cases of ld among hispanics were from new york or new jersey ( table 2 ) . among counties with at least 75 estimated ld cases among hispanics during the study period , highest incidence among hispanics occurred in columbia county , new york ( 170.4 cases/100,000 persons ) ; sussex county , new jersey ( 111.4/100,000 ) ; and hunterdon county , new jersey ( 106.3/100,000 ) . after correcting for missing ethnicity data . calculated as follows : ( no . reported cases)/x = ( % with ethnicity information)/100 , where x is the weighted number of cases . incidence calculated as number of annual estimated cases in county/100,000 hispanic residents in county . only counties with a substantial number of cases were included in this comparison . overall , the epidemiology of ld among hispanics was similar to that among non - hispanics : bimodal age distribution , slight predilection in males , and clustering in states to which ld is highly endemic were apparent ( 10 ) . most notably , hispanics with ld were significantly more likely than non - hispanics with ld to have signs of disseminated infection and symptom onset during fall months . although the overall incidence of ld in hispanics was lower than that in non - hispanics , additional research is needed to determine the reasons underlying these differences and the extent of any ld underdiagnosis in the hispanic population . inadequate healthcare access , language barriers , and lack of ld awareness could cause both underdiagnosis and delays in diagnosis in the hispanic population . during 20092013 , a total of 41.5% of hispanics lacked health insurance , compared with 15.1% of non - hispanic whites ; 15.5% of hispanics described delay in or nonreceipt of medical care because of cost ( 11 ) . furthermore , whether the predilection toward symptom onset in the fall months for hispanics results from delays in medical care or other factors , such as seasonal outdoor labor patterns , is unclear . lastly , because a larger proportion of hispanics than the overall us population live in urban areas ( 12 ) , the risk for ld might be differentially diluted in hispanics . first , we had to exclude more than half of reported ld cases because of missing ethnicity data . although we have no reason to believe that case reports with missing ethnicity data differed otherwise from those included in this study , we can not exclude this possibility . finally , surveillance data are limited by underreporting and reporting bias , which might differ by state and between hispanic and non - hispanic populations . although some educational materials about prevention of tickborne diseases have been translated to spanish ( 13,14 ) , additional translations and modifications to address cultural differences would be helpful . furthermore , targeted educational campaigns could enhance use of these materials and improve the reach , retention , and overall impact of prevention education . direct and more in - depth assessments regarding prevention practices , knowledge , and ld epidemiology on local and national scales will further the understanding of ld risk in this population and guide future targeted prevention and education efforts .

background : treatment of elbow dislocation with irreparable radial head fracture needs replacement of radial head to achieve stability of elbow . an alternate method in cases of elbow dislocation with radial head fracture can be resection of radial head with repair of medial collateral ligament . we report a retrospective analysis of cases of elbow dislocation with irreparable radial head treated by excision head of radius and repair of mcl.materials and methods : nine patients of elbow dislocation with associated irreparable fractures of the head of the radius were included in this analysis ( 6 f:3 m , age : 35 - 47 years ) . radial head excision was done through the lateral approach and mcl was sutured using no 3 ethibond using medial approach . above elbow plaster was given for 6 weeks and gradual mobilization was done thereafter . all patients were assessed at final followup using mayo elbow performance score ( meps).results : mean followup was 19.55 7.12 months ( range 14 - 36 months ) . there was no extension deficit when compared to opposite side with mean range of flexion of 138.8 6.97 ( range 130 -145 ) . mean pronation was 87.7 4.4 ( range 80 - 90 ) and mean supination was 87.7 4.62 ( range 80 - 90 ) . the mean meps was 98.8 3.33 ( range 90 - 100 ) . no patient had pain , sensory complaints , subluxation or redislocation . all were able to carry out their daily activities without disability.conclusion:radial head excision with mcl repair is an acceptable option for treatment of patients with elbow dislocation and irreparable radial head fracture . hispanics comprise a growing portion of the us population and might have distinct risk factors for tickborne diseases . during 20002013 , a total of 5,473 lyme disease cases were reported among hispanics through national surveillance . hispanics were more likely than non - hispanics to have signs of disseminated infection and onset during fall months .

chronic cardiovascular diseases ( cvds ) are considered the leading cause of mortality worldwide ( 1,2 ) . mortalities by cvds occur mainly due to coronary heart disease ( chd ) , stroke , rheumatic heart , and myocardial infarction ( mi ) . approximately 80% of these deaths occur in low and middle income families and in poor countries ( 3 ) . it is estimated by who that approximately 20 million individuals are likely to succumb to cvds in the year 2015 ( 4 ) . in addition , non - communicable diseases are to account for over 75% of mortalities worldwide ( 1 ) . infectious diseases , tuberculosis , and malaria followed by nutritional disorders are the primary cause of such mortalities ( 5 ) . cvds are also the largest contributor to global mortality of heart patients ( 3,6 ) . the epidemiology of heart diseases has been described by many researchers in the literature . for the epidemiological studies of heart diseases , a significant percentage of cvd mortalities occur in younger individuals and children in developing nations due to malnutrition , low income , poverty , unemployment , excessive alcohol consumption , tobacco smoking , car accidents , and other factors ( 5 ) . approximately 41% individuals die between the age of 35 and 64 years in south africa , 35% in india , 28% in brazil , 12% in the usa , and 9% in portugal ( 7 ) . the median age of heart attack and first stroke and death from ischemic heart disease ( ihd ) as well as acute myocardial infarction ( ami ) are very important ( 3 ) . hypertension , high blood pressure ( hbp ) , high blood cholesterol , obesity , diabetes , reduced physical activity , psychological stress and other factors are also responsible for cvds . the historical trends in cv mortality and tobacco use in north america increased between 1900 and 1990 ( 8) . in the uk , a 38-year follow - up study of men showed that baseline differences in tobacco use , hbp pressure , and cholesterol were associated with a 10- to 15-year shorter life expectancy ( 9 ) . the rise in cigarette smoking in young individuals indicates increased tobacco - related mortalities in many countries ( 10 ) . by the year 2030 , who ( 2008 ) projects that over 80% of tobacco - related deaths are to occur in young people in developing countries . currently , malnutrition and obesity occur within the same population in many low and middle income families ( 11,12 ) . malnutrition is common in many low and middle income countries of africa , latin america , and south asia ( 7 ) . the global prevalence of overweight in children aged 517 years is 10% , varying from under 2% in sub - saharan africa to over 30% in the usa ( 14 ) . the epidemiological research of cvds suggests that dietary changes are associated with nutritional transition , specifically the increasing consumption of energy - dense diets high in fats , oils , sodium , and sugars , which contribute to an increase in cvd incidence in low and middle income families ( 15 ) . the kilo caloric intake from selected food groups in china , india , mexico , egypt , and south africa from 1980 to 1982 were compared to the kilo caloric intake in 2001 to 2003 using data from the food and agriculture organization of the united nations statistical database ( faostat ) ( 16 ) . in china , total lcd ( low - calorie diet ) increased from 2,327 to 2,940 kcal , and meat consumption increased by over 246% from 1980 to 2003 . there was also a significant increase in the intake of oils in china , with three types of oils increasing over 100% : palm oil ( + 640% ) , soybean oil ( + 635% ) , and vegetable oils ( + 259% ) . on the positive side , the intake of fruits and vegetables also increased by 600 and 367% , respectively ( 16 ) . between 1980 and 2003 , there has been an increase in meat as well as greasy food consumption in china , as well as increased use of tropical oils ( which is associated with high cholesterol and not healthy for heart ) in preparing food . similarly , in india , mexico , south africa and other nations the elevated kcal intake of palm oil in the diets was reported ( 16 ) . the causes of the decline in cvds in developed countries offer potential lessons for achieving similar results in developing countries . the major risk factors for cvds have been reported for childhood and adolescence ( 1720 ) . these include use of tobacco , diets and physical activities , overweight and obesity , and adverse childhood experiences . poor social life circumstances in childhood have also been linked to cvds later in life in different cohort studies conducted in the usa and europe ( 21,22 ) . a cohort study of 20,040 individuals four measures of health were examined : smoking , low physical activity , low plasma vitamin c levels ( used as a proxy for fruit and vegetable intake ) , and not drinking alcohol in moderation ( abstaining from alcohol or consuming more than 14 drinks per week ) and the results indicated that one or more of the abovementioned risk factors alone or in combination with other risk factors constituted a > 2-fold increase in stroke incidence ( 23 ) . this is consistent with prior findings in large cohort studies on men and women in the usa whereby a healthy diet and lifestyle of not smoking , regular exercise , moderate alcohol consumption , and not being overweight was associated with an almost 80% lower risk of ihd compared to having none of the abovementioned healthy lifestyle components ( 24 ) . the major risk factors for cvd in young individuals or adults include fats , abnormal blood lipids , tobacco smoking , alcohol , abdominal obesity , psychosocial factors , hypertension , and diabetes . there is strong evidence that , excessive salt and sodium intake significantly increases cvd risk and that reduction in sodium intake decreases incidence of cvds ( 25 ) . three major dietary patterns were identified : i ) oriental ( high intake of tofu and soya ; ii ) western ( high in fried foods , salty snacks , eggs , and meat ) ; and iii ) prudent ( high in fruits and vegetables ) . the results showed the western dietary pattern was associated with an increased risk of chd in animals and humans globally , whereas the prudent pattern with fruits and vegetables was associated with a lower risk of heart diseases in children , young and adult individuals ( 26 ) . increased daily consumption of alcohol has been reported as a great risk factor of chd in individuals of all ages in low and middle income groups ( 27 ) . the who estimates that the harmful use of alcohol was responsible for 3.8% deaths and 4.5% of the global burden of heart diseases in 2004 ( 3 ) . in the past few decades , consumption of alcohol has increased in countries undergoing nutritional transition , such as india and china , and russia , where it contributes significantly to overall mortality among men ( who expert committee , 2007 ) ( 2 ) . excessive alcohol intake is associated with increased risk for hypertension , stroke , coronary artery disease , and other forms of cvds ; however , evidence conducted in various populations suggests that light to moderate intake of alcohol may reduce the risk of chd ( 2 ) . the relationship between one drink per day for women and no more than two drinks per day for men constitute moderate drinking for health of heart ( 28 ) . physical exercise is also important for the health of heart as who and the food and agriculture organization ( fao ) suggest the importance of physical activity as a key determinant of obesity , cvd , and diabetes ( 29 ) . obesity or overweight issues , reported from developed and developing countries are considered risks of cvds ( 30 ) . malnutrition remains highly prevalent compared to overweight and obesity , especially among women and children ( 31 ) . the intake of cereals is also important in china , where 58% of total calorie intake is consumed compared to meat , 13% , and cooking oils , 17% ( 32 ) . the national data from the office of national statistics indicates that cereals and related products account for 31% of calories with other major categories including meat , 15% ; milk and related products , 19% ; and beverages , 10% ( 33 ) . in a study on hbp it was found that approximately 54% of individuals experience stroke , 47% ihd , 75% hypertensive disease and 25% other cvds that were attributable to hypertension . approximately 7.6 million individuals succumb to hypertension , with 13.5% of the total number of annual global deaths being attributable to hbp ( 34 ) . it is estimated that the current age - standardized prevalence rate of hypertension is 17.7% , which amounts to approximately 177 million individuals , while 20% of individuals in china may succumb to hbp due to a high intake of salt ( 35,36 ) . the framingham study showed a connection between hypercholesterolemia and increased risk of chd in 1960 with the finding that lower levels of high - density lipoprotein ( hdl ) cholesterol as well as elevated levels of low - density lipoprotein ( ldl ) cholesterol were associated with increased risk of chd ( 37,38 ) . the results of several randomized controlled trial studies have shown that the reduction of ldl cholesterol , both in primary and secondary prevention , is associated with a lower incidence of chds ( 4143 ) . the interheart study found that abnormal blood lipids were the most important risk factor for mi , by odds ratio in all global regions ( 44 ) . elevated cholesterol levels were the third leading risk factor for the worldwide mortality of young and elderly individuals , after hypertension and smoking ( 45 ) . the risk of cvd associated with diabetes is even greater in women and younger individuals . having diabetes may be equivalent to aging by at least 15 years with regard to the clinical manifestations of cvd ( 46,47 ) . diabetes is associated with serious health consequences and is a major risk factor for chd and stroke . depression and depressive symptoms are also associated with behaviors that increase cvd risk ( 48 ) . anxiety , anger , hostility , chronic and acute stress , and lack of social support are factors associated with increased cvd morbidity and mortality . the acute stress from traumatic injury , death of a relative , earthquakes , terrorist attacks or other factors have been associated with significant temporal increases in the incidence of mi in young or adults ( 49 ) . research has also shown that air pollution and respiratory problems have adverse health outcomes associated with lung - heart or cardiovascular effects ( 50 ) . the burning of coal and wood used for cooking is also a significant contributor of air pollution in developing countries . the epidemiological studies of heart diseases have also shown that long - term exposure to smoke can increase the risk of cvd ( 51 ) . molecular genetics and advanced statistical methods , used for the study of heart diseases and proteomics methods can lead to potential biomarkers that can identify risk of cvds , which in turn , can improve the prediction of acute cardiovascular events ( 52 ) . pharmacogenetics can be useful in the development of more effective medications , and the management of cvd in children , young and adult population . rheumatic heart disease ( rhd ) , chagas disease , and infectious pericarditis and cardiomyopathies continue to cause a substantial burden of disease and death in some low and middle income countries ( 53 ) . a better epidemiological understanding of infectious forms of cvd in developing countries , and efforts to improve health care are needed to prevent and treat these diseases . infectious diseases such as hiv / aids , and tuberculosis ( tb ) , and other diseases such as malaria , typhoid and diabetes are also important for their association with cvd ( 53 ) . in order to study cvds in the developing and developed nations , efficient epidemiological programs should be designed . chd accounts for the maximum proportion of cvds for which risk factors are hypertension , smoking , diabetes , high cholesterol , higher level of ldl , obesity and overweight causing death of heart patients . assessment of short- or long - term risk prediction algorithms is useful to identify patients that can benefit most from risk factor interventions . the evaluation of new risk factors and screening for subclinical atherosclerosis are imperative in the identification of individuals that are at the highest risk of cvd . the prevention of chds must focus on identifying and managing risk factors in individual cases as well as the population through primordial , primary , and secondary prevention . epidemiological studies have provided a hypothesis based on clinical trials for chd and the efficacy of risk - factor interventions , which are the basis of prevention of heart diseases ( 55 ) . further investigations on screening and intervention strategies is important to determine the effect on the prevention of chds and cvds . recent advances in the epidemiology , pathogenesis and prognosis of acute heart failure and cardiomyopathy have been described from africa ( 56 ) . a large prospective registry showed that acute decompensated heart failure is caused by hypertension , cardiomyopathy and rhd in 90% of cases . this is a pattern that is in contrast with the dominance of coronary artery disease in north america and europe ( 56 ) . acute heart failure is a disease of young individuals with a mean age of 52 years and occurs equally in men and women . this observation was associated with high mortality at 6 months ( approximately 18% ) , but was similar to that observed in non - african heart failure registries , suggesting that heart failure was a dire prognosis globally , regardless of the aetiology . investigations into the molecular genetics of dilated cardiomyopathy , hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy in populations in africa are consistent with observations elsewhere ( 56 ) . in addition , the study of genotype - phenotype correlations in a large number of cardiomyopathy individuals is due to the same mutation ( 56 ) . thus the use of proven treatments , and the control of hypertension in various populations should be increased . the american heart association ( aha ) has recommended following a mediterranean diet for heart patients ( 57 ) , in which more fruits and vegetables are included . at least 16 countries border the mediterranean sea and their diets vary among these countries . there are numerous differences in culture , ethnic background , religion , economy and agricultural production that result in a difference in mediterranean diets . the common mediterranean dietary pattern has these characteristics : high consumption of fruits , vegetables , bread and other cereals , potatoes , beans , nuts and seeds , olive oil which is an important source of monounsaturated fats , dairy products , fish and poultry consumed in low to moderate amounts , and a modest amount of red meat ( 57 ) . in such a diet , eggs are consumed 04 times a week and wine in low - to - moderate amounts . individuals who adhere to an average mediterranean diet usually eat less saturated fats than those who consume an average american diet . it is known that over 50% of fat calories in a mediterranean diet are derived from monounsaturated fats , mainly from olive oil . monounsaturated fats do not raise the level of blood cholesterol in the same way as saturated fat . the incidence of heart disease in mediterranean countries is lower than that in the united states , as are mortality rates . diet as well as lifestyle factors , such as more physical activity and extended social support systems may also be important in heart diseases . in recent advances in preventive cardiology and lifestyle medicine , public policy approaches to the prevention of heart disease and stroke have been described ( 58 ) . the global epidemiology of heart disease and stroke in the 20th century showed marked variations in cause - specific mortality rates between developed and developing countries due to trends of social and political origins of cardiovascular risk . thus , governments and the private sectors are attempting to educate the public and coordinate public efforts to retard the global epidemic of heart disease and stroke ( 59 ) . the osaka declaration has emphasized factors for the health sector that serve as impediments to heart health ( 60 ) . success in the treatment of heart diseases and conditions of mi , valvular heart disease , and arrhythmias has improved patient survival . in the elderly , congestive heart failure is the leading cause for admission to hospitals in the united states , which is extremely expensive for health care ( 61 ) . african - americans , aged 4565 years , have a 70% higher rate of heart failure as shown in the census bureau and the centers for disease control and prevention , with a mortality rate 2.5-fold higher than that of the caucasian population . the hispanic / latino population has a higher incidence of heart failure than the caucasian non - hispanic population . however , more population - based studies are needed to estimate the prevalence of heart failure in the hispanic population ( 62 ) . useful sites for heart disease research include : the american college of cardiology ( http://www.acc.org/ ) , for up to date heart failure patient 's information ( http://www.uptodate.com/home ) ; national library of medicine , ( https://www.nlm.nih.gov/ ) ; national heart , lung , and blood institute ( http://www.nhlbi.nih.gov/ ) ; european society of cardiology ( http://www.escardio.org/ ) ; and heart failure online , http://www.heartfailure.org/. recent investigations on cvds in europe for 2014 have shown that , epidemiological aspects have been updated ( 63 ) . cvds and in particular chd and stroke have been previously demonstrated in european countries ( 63 ) . cvds cause more deaths among europeans than other countries , and cause more than twice as many deaths as cancer . it has also been reported that for some eastern european countries , including russia and the ukraine , the mortality rate for chd in 55- to 60-year - old individuals is greater than the equivalent rate in france . the epidemiology of cvd in the uk in 2014 was described with regard to mortality , prevalence and treatment costs ( 64 ) . in the year 2012 , cvd was the most common cause of mortality in the uk for women at 28% of all female deaths , but not for men , whereas cancer is currently the most common cause of mortality at 32% of all male deaths . mortality from cvd varies widely throughout the uk , with the highest age - standardized cvd death rates in scotland ( 347/100,000 ) and the north of england ( 320/100,000 in the north west ) . the prevalence of chds is also highest in the north of england ( 4.5% in the north east ) and scotland ( 4.3% ) . thus , primary and secondary prevention measures are important to reduce the burden of cvd and inequalities in cvd mortality rate and prevalence . the epidemiology of heart failure has been reported and it has been suggested that chd is prevalent ( 65 ) . the excessive use of tobacco smoking , alcohol consumption and other factors are also responsible for heart diseases . the present review focused on recent epidemiological aspects of cvd and chd as well as the benefits of a mediterranean diet for heart patients and its usefulness in preventing cardiovascular disease . in general , there are several risk factors such as high blood cholesterol , high blood pressure , obesity and family history that are associated with cvd and chd results obtained from several studies support the positive effects of the mediterranean diet for the primary prevention of cardiovascular disease . the mediterranean diet is characterized by a high intake of olive oil , fruit , vegetables , and cereals ; a moderate intake of fish and poultry , and a low intake of dairy products , red meat , processed meats , and sweets , as well as wine in moderation , consumed with meals . future studies may shed more light on the notable effects of the mediterranean diet on the prevention of cardiovascular disease . metastases to the spleen from various sites are a rare occurrence and usually occur in the presence of widely disseminated disease . previously documented sites of primary disease include the ovaries , melanoma , breast , lung and the colon and are typically associated with high disease burden . splenic metastases from the colon involving low levels of disease burden are rare in the literature and to date , there have been only two documented cases of metastases to the spleen originating from a caecal tumour . a 60 year old male with no significant past medical history presented to the emergency department with a three week history of lower abdominal pain , nausea and a palpable mass in the right iliac fossa . computerised tomography ( ct ) of the abdomen and pelvis demonstrated a large circumferential obstructing tumour of the caecum without evidence of distant metastases ( fig . the patient was prepared for surgery , consisting of a right hemicolectomy with an uncomplicated postoperative course . histology from the excised specimen revealed a poorly differentiated adenocarcinoma with signet ring cell permeation through the muscularis propria . lymph node analysis comprised twenty one lymph nodes , all noted to contain metastatic spread . the presenting tumour was therefore classified pathologically as stage iii caecal adenocarcinoma ( pt3n2m0 ) . carcinoembryonic antigen measured prior to surgery was recorded within normal limits ( 3 ng / l ) . two months post surgical resection , the patient underwent adjuvant chemotherapy utilising the folfox protocol as discussed at the multidisciplinary conference . he received 12 cycles of chemotherapy however the dose of oxaliplatin was reduced after cycle six due to development of thrombocytopaenia and a slight rise in cea level , both of which were attributed to the chemotherapeutic agent ( table 1 ) . the patient was then entered into a standard programme of routine surveillance consisting of regular clinical reviews , interval measurement of cea levels , colonoscopy and yearly ct of thorax , abdomen and pelvis . although surveillance studies proved negative initially for disease recurrence , a routine ct thorax , abdomen and pelvis at twenty three months post initial presentation revealed left sided para aortic lymphadenopathy and two suspicious splenic lesions measuring 2.7 2.5 cm and 2.4 2.2 cm respectively which were not present on imaging , performed twelve months previously ( fig . 2 a and b ) . the splenic lesions were biopsied under ultrasound guidance using an 18 gauge tru cut needle without complication ( fig . subsequent histology of the biopsied lesions revealed normal splenic parenchyma infiltrated by poorly differentiated adenocarcinoma with abundant signet ring cells , morphologically identical to the initial presentation with an obstructing caecal tumour ( fig . the disease although of low burden and primarily localised to the spleen was associated with the presence of suspicious para aortic lymphadenopathy prompting chemotherapy rather than operative intervention . the oncology team advised further systemic chemotherapy comprising 5 fluorouracil , oxaliplatin and avastin . the patient remains well on chemotherapy and further imaging studies are planned in 4 months time to assess response to treatment . metastases to the spleen from any primary tumour are rare despite the highly vascular nature of the organ and it 's proximity to many potential primary sites including the pancreas , stomach , lung , breast and the colon . a number of theories have been put forward to explain the hostile nature of the spleen preventing tumour implantation and proliferation of neoplastic cells . anatomical features thought to play a role include the sharp angle at the origin of the splenic artery with the coeliac axis , the rhythmic contraction of splenic sinusoids and the scarcity of lymphatic vessels extending to the intrasplenic parenchyma . immunological factors considered to negatively impact on the rate of malignant cell implantation include the presence of abundant kupffer cells , immunoglobin synthesis and opsonin production . in most of the reported cases of colorectal metastases to the spleen , the primary tumour is located on the left side of the colon or in the rectum . this distribution of colorectal metastases favours the theory that neoplastic cells reach the spleen via retrograde dissemination through the inferior mesenteric vein . to the best of our knowledge , there have been only two previous cases of caecal adenocarcinoma metastasising to the spleen recorded in the literature without infiltration of other solid organs . furthermore , there have only been 4 additional cases of right sided colonic cancer ( all ascending colon ) with metastases to the spleen recorded . given the additional presence of left sided para aortic lymphadenopathy in our patient , this could possibly indicate tumour spread via the lymphatic system . it is not routinely used as a screening tool as it lacks sensitivity and specificity . however it is widely used as part of surveillance programmes to monitor for disease recurrence in conjunction with colonoscopy , imaging and follow up clinic appointments . it has been shown that in 3040% of cases of disease recurrence there is no corresponding rise in cea levels . furthermore in cases where cea levels were recorded as normal at initial presentation , it was reported that in cases of recurrence , a corresponding elevation in cea is only observed in 27% of cases . in one large study , it was noted that cea monitoring did not result in improved survival . this brings into question the value of interval cea monitoring as part of surveillance programmes post resection for colorectal cancers . historically the technique of percutaneous splenic biopsy was not widely performed due to the perceived high complication rates mostly involving haemorrhage . with the advent of improved imaging modalities , splenic biopsies can now be performed relatively safely using either ultrasound or ct guided techniques . the differential of a solid splenic lesion includes metastases , an infarct or a splenic abscess . adequate collection of targeted tissue suitable for histological analysis has been recorded at 88% in one series . complication rates were found to be directly proportional to the gauge of needle used in the procedure . given the limited number of cases recorded in the literature , the optimal treatment for splenic metastases from a colorectal primary is not known . in cases of isolated splenic metastases from a colonic source , splenectomy is preferred to chemo / radio therapy . in the literature , this has been performed by both the traditional open technique and more recently laparoscopically . to date disease free intervals and survival are reported to be similar for both techniques . chemotherapy is currently favoured in circumstances where disease is recorded in other sites including the spleen . the current data available is too small to draw any definitive conclusions regarding the optimal treatment for splenic metastases in the context of low disease burden . firstly , multimodal imaging is important in the context of asymptomatic patients and especially given the limited sensitivity of cea meaurements . second , modern image guided biopsy techniques facilitate accurate and safe percutaneous biopsy of suspicious splenic lesions . third , further research is necessary to determine the optimal treatment of splenic metastases from a colonic source with a low disease burden . written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request . kevin barry helped in the final review of article prior to submission including editing and layout .

cardiovascular diseases ( cvds ) are the leading cause of mortality worldwide . coronary heart disease ( chd ) is the main cause of mortality in heart patients following stroke , rheumatic heart disease and myocardial infarctions . approximately 80% of individuals succumb to cvds , due to poor living conditions in low and middle income families and malnutrition . infectious diseases , human immunodeficiency , tuberculosis , malaria , high blood pressure or hypertension , obesity and overweight , and nutritional disorders including smoking , excessive alcohol consumption , high salt and sugar intake , as well as other factors are responsible for cvds and chds in young as well as elderly individuals . the focus of the present review are recent epidemiological aspects of cvd and chd as well as the usefulness of a mediterranean diet for heart patients and the prevention of heart diseases . introductionthe spleen is a highly vascular organ and is in close proximity to many potential primary sites such as the stomach , breast , pancreas and colon . it is however an unusual site for metastatic disease . the reasons for this are not fully understood at the present time . a number of hypotheses have been postulated . definitive diagnosis and subsequent treatment of metastatic disease to the spleen presents a number of challenges for the surgeon and the wider multi disciplinary team.presentation of casea 60 year old male presented with a three week history of lower abdominal pain , distension , nausea and a palpable mass in the right iliac fossa . imaging revealed a large circumferential caecal mass consistent with malignancy with secondary small bowel obstruction . the patient underwent an emergency right hemicolectomy and was subsequently treated with systemic chemotherapy for lymph node positive caecal adenocarcinoma . two years following initial presentation , two suspicious lesions were noted within the spleen during routine surveillance imaging with computerised tomography of the thorax , abdomen and pelvis . of note , one month prior to this the patient had a normal surveillance colonoscopy performed with multiple interval carcinoembryonic antigen ( cea ) levels recorded within the normal range . image guided splenic biopsy and subsequent histology confirmed metastatic caecal adenocarcinoma.discussionsplenic metastases from any source including the colon are a rare phenomenon.conclusionthis case questions the value of routine post operative cea monitoring , underlines the importance of multimodal pathways of surveillance and highlights recent advances in image guided splenic biopsy techniques .

recent advances in repair of thoracoabdominal aortic aneurysms ( taaa ) allow one to choose between three therapeutic options : conventional open surgery , a hybrid open - endovascular procedure or endovascular repair . despite significant progress in operative technique and procedural anesthesia , a conventional open surgical repair of taaa is still accompanied by significant morbidity and mortality . a number of large studies dealing with outcomes following open taaa surgical treatment indicate that the 30-day mortality of elective cases ranges between 5% and 19% , while one - year mortality increases to even 30% [ 1 , 2 ] . however , only experienced high - volume vascular centers can treat taaa surgically with a mortality rate below 10% . in high - risk patients , hybrid procedures combining extra - anatomic bypasses and endovascular techniques with branched and fenestrated aortic stent grafts have been used to reduce the risk of conventional taaa with the repair of visceral arteries . in patients with crawford type iv taaa , employment of fenestrated aortic stent grafts has been proved to provide better early results than open surgical procedures . endovascular repairs in patients with taaa are limited so far to anatomical factors , such as ostial stenosis , severe vessel angulations and tortuosity as well as narrow iliac , renal and visceral arteries . other limitations to the common use of fenestrated stent grafts include high costs and the time delay between qualification and manufacture of the tailored grafts . today , there are only a few studies in the literature reporting application of fenestrated stent grafts in the treatment of complex abdominal aortic aneurysms involving four visceral vessels . we describe a case of a high - risk patient with taaa involving the visceral vessels in which the endovascular technique was chosen as the most appropriate therapeutic option . a 56-year - old man was diagnosed with asymptomatic type iv taaa with a maximal diameter of 55 mm ( fig . a number of risk factors that could increase predicted mortality and morbidity rate such as hypertension , type ii diabetes mellitus treated with insulin , symptomatic coronary artery disease ( canadian cardiovascular society , ccs class ii ) and severe chronic obstructive pulmonary disease ( fev1 below 1l ) were diagnosed . the pre - operative computed tomography angiography ( cta ) that confined the whole descending aorta from the arch to bifurcation and further to the common femoral arteries demonstrated a type iv taaa and patent normal celiac trunk as well as the superior mesenteric artery originating from the aneurysm . ( a ) ct scan with thoracic aorta ( 24 mm in diameter ) above an aneurysm . ( b ) ct scan at the level of the aneurysm with maximal diameter of 55 mm . ( c ) ct scan with abdominal aorta below the aneurysm the stent graft was customized after meticulous calculations such as length and diameter at the proximal and distal implantation sites within normal aortic segments ( fig . 1 ) , diameters of both iliac and femoral arteries , all angulations , the ostial diameter of the renal arteries , the superior mesenteric artery and the celiac trunk as well as distances between these vessels were done . the stent graft diameter at both ends was 28 mm , length was 178 mm , and it had four holes ( two 8 mm and the other two 6 mm in diameter ) . they were exposed surgically through longitudinal skin incisions approximately 5 cm in length in both groins . after the arteries were dissected free intravenous heparin then a purse - string stitch with 5 - 0 monofilament polypropylene non - absorbable suture was placed on the right femoral artery and an endovascular graft was delivered using the seldinger technique ( the sheath size was 20 fr ) . after an appropriate position was achieved in the region of the aortic aneurysm , a stent graft was deployed . then the superior mesenteric and both renal arteries were successfully cannulated from the left common femoral artery . the covered advanta v12 stents ( atrium medical corporation , nh , usa ) were placed to secure the interface between the stent graft fenestrations and superior mesenteric and both renal arteries orifices . the sizes of the renal and superior mesenteric stents were 6 mm 22 mm and 8 mm 38 mm , respectively . afterwards , the celiac trunk was cannulated from the left brachial artery open surgical approach ( as described above ) and was stented with a covered 8 mm 60 mm fluency stent ( bard peripheral vascular , tempe , ar , usa ) ( fig . consecutive steps of endovascular procedure under guidance of digital subtraction angiography ( dsa ) complete exclusion of the aneurysm with no endoleak and with preservation of flow in the celiac trunk , the superior mesenteric artery and both renal arteries was confirmed . the sites of cannulation of surgically exposed peripheral arteries ( both femoral and left brachial ) were fixed with previously placed purse - string stitches and the wounds were closed with layers with absorbable sutures . the total stent graft implantation time was 250 minutes and the fluoroscopy time was 54 minutes . follow - up cta at 6 months after endovascular repair showed successful sealing with complete exclusion of the aneurysm and proper perfusion in the four target vessels ( fig . properly deployed stent graft in the abdominal aorta and stents in the proximal segments of the visceral vessels ( celiac trunk , upper mesenteric and both renal arteries ) pararenal and taaa repair by fenestrated or branched stent grafts as a minimally invasive technique seems to offer good short- and medium - term results in comparison to conventional open surgery . a conventional operation for taaa requires extensive exposure that requires opening not only the abdominal but also not uncommonly the pleural cavity . this surgical excess itself is associated with at least moderate risk for the development of serious postoperative complications . moreover , rehabilitation after open surgery is long , and mobilization of the patient is only possible a few days after the operation . our patient was mobilized and rehabilitation was initiated on the second day after surgery . additionally , we are convinced it enabled us to avoid many possible complications related to mechanical ventilation of an individual with advanced chronic obstructive pulmonary disease . thus , the indications for endovascular repair of type iv taaa are high - risk patients ( e.g. elderly subjects , individuals with several comorbidities ) with favorable anatomy that makes it possible to manufacture the customized fenestrated stent grafts . adequate visualization of all visceral branches and proper sizing of the fenestrated stent grafts are of crucial importance to ensure success in the endovascular treatment of taaa . these devices were manufactured on the basis of cta data with bespoke fenestrations at the level of the orifices of four target vessels . considerable experience in endovascular grafting is necessary , especially during deployment of the fenestrated graft in an accurate position and fixation of the graft fenestrations with renal and visceral orifices by implantation covered stents . stenting of the fenestrated graft to the renal and visceral orifices as well as the branched graft have a favorable impact on preservation of blood flow through the graft - ostial junctions and target arteries . additionally , these covered stents placed in the graft - ostial tracts may also prevent graft migration and endoleaks as well as renal and visceral artery occlusions . the significant injury during open surgery especially in high - risk patients leads to the development of serious perioperative complications . meanwhile , during endovascular repair there are real chances to avoid or at least significantly decrease the rate of organ complications . despite the good outcome of endovascular repair of taaa postoperative complications occurred in 11% to 14% of patients and included heart failure or myocardial infarction ( 5.5% to 6.5% ) , stroke ( 1.4% to 2.5% ) , respiratory insufficiency ( 3.8% to 6.8% ) , neurological events such as paraplegia or paraparesis ( 1.1% to 2.7% ) and renal dysfunction ( 1.4% ) . in the perioperative period prevalence of endoleaks the early occurrence of renal dysfunction may be provoked by the use of a large volume of contrast agents predisposing to contrast - induced nephropathy or embolization of the renal arteries during deployment of the stent graft . moreover , the use of fenestrated and branched stent grafts was proved to be beneficial , particularly in patients with preexisting severe cardiovascular , pulmonary , neurological or renal dysfunction . for these reasons , even complex endovascular therapy with fenestrated stent grafts may offer shorter hospitalization and faster post - procedural rehabilitation . our uncomplicated case demonstrated at 6 months after the procedure complete aneurysm exclusion with 18 mm reductions in aneurysm sac diameter and preserved perfusion in the stent graft and four visceral arteries . thus , complex and diffuse aortic pathologies such as thoracoabdominal aneurysms may be treated successfully with fenestrated stent grafts . however , appropriate patient selection , proper graft design and a multidisciplinary team experienced in endovascular procedures are of paramount importance in successful treatment of this particular group of patients . renal cell carcinoma ( rcc ) may metastasize to any site of the body , but clinically evident metastatic intestinal involvement by rcc is extremely rare . to our knowledge , simultaneous duodenal and colonic metastases have not been reported in the english literature . we report a case of pathologically proven simultaneous duodenal and ascending colonic metastases about four years after a left nephrectomy for rcc . a 76-year - old female patient who had undergone a left radical nephrectomy 4 years previously for rcc ( mixed clear and granular cell type , tnm stage iii ) presented with a 1-month history of dyspepsia , lethargy and pain in the right upper abdomen . her blood pressure was 110/70 mmhg , pulse rate 80/min , respiration rate 22/min and body temperature 36.8c . abdominal examination revealed slight tenderness but normal peristalsis ; however , a movable mass was palpated in the right upper abdomen . the hematocrit was 28.1% and the white cell count was 9700/mm with 77% polymorphonuclear cells and 14% lymphocytes . serum sodium was 134 meq / l , potassium 3.5 meq / l , chloride 101 meq / l and calcium 9.5 mg / l . the result of liver function tests were as follows : total protein 6.5 g / dl , albumin 3.5 g / dl , cholesterol 165 mg / l , bilirubin 0.6 mg / dl , alkaline phosphatase 105 iu / l , ast 28 iu / l and alt 21 iu / l . tumor marker levels , such as carcinoembryonic antigen ( cea ) , carbohydrate antigen 19 - 9 ( ca19 - 9 ) and -fetoprotein ( afp ) , were within normal limits . an abdominopelvic ct scan showed circumferential wall thickening with high enhancement at the second portion of the duodenum and additional enhancement of an irregular protruding mass into the lumen of the ascending colon with multiple lymphadenopathy in the aortocaval area ( figure 1 ) . a gastroscopy showed a large , irregular multi - lobed , partially necrotic and ulcerative protruding mass nearly obstructing the second portion of the duodenum . a colonoscopy revealed a polypoid , nodular mass in the ascending colon , which was purplish in color , had sharp margins and was pliable ( figure 2 ) . microscopic findings of biopsy specimens showed features identical to those of the renal cell carcinoma which was resected 4 years earlier in this patient and immunohistochemical stainings for vimentin was positive ( figure 3 ) . interferon was given at a dose of 2 million iu per square meter three times a week . however , interferon therapy was discontinued after three months due to anemia , anorexia and general weakness . the clinical course of the disease ranges from months to several decades and even spontaneous regression has been documented . approximately 25% of patients with rcc have metastatic disease at the time of diagnosis . the delayed occurrence ( as late as 31 years after a nephrectomy ) of metastatic rcc is well known . solitary metachronous metastases from rcc are rare ; however , they can occur very late in the course of the disease . renal cell carcinoma may metastasize to almost every organ of the body , but 95% of the metastatic lesions involve the lung , lymph nodes , liver , bone , adrenal glands and the opposite kidney . however , there are scattered reports in the literature of clinically evident intestinal metastasis from rcc . to the best of our knowledge , a secondary tumor involving the intestinal tract may be caused by direct extension , lymphatic spread , peritoneal or hematogenous dissemination . in the present case , duodenal and colonic metastases are not generalized carcinomatosis because any other metastasis is not occurred in common metastatic sites and duodenal and ascending colonic involvement are remote from a primary cancer of the left kidney . hypervascularity of the present tumor , as suggested by a ct scan , gastroscopy and colonoscopy , was compatible with the characteristics of metastatic renal cell carcinoma . also , positive immuno - histochemical stain for vimentin elucidated that the duodenal and colonic masses are compatible with carcinoma rather than adenocarcinoma . surgical excision of the local recurrence is the best procedure for therapy , but this can be radical only when the recurrence can be completely excised . chemotherapies , including hormonal and interferon therapies , are effective in some patients with metastatic renal cell carcinoma . in our case although interferon treatment was used to treat metastatic rcc , it is not easy to evaluate its effectiveness because of premature discontinuance of the treatment . in our opinion , endoscopists should consider the possibility of intestinal metastasis of rcc when endoscopic and ct studies show a hypervascular mass in a patient with a previous history of rcc .

conventional open surgical repair of thoracoabdominal aortic aneurysm ( taaa ) is associated with high perioperative mortality and morbidity risk . our report of successful treatment of a 56-year - old patient with taaa involving all visceral arteries and with many comorbidities with a fenestrated stent graft supports its application in high - risk taaa patients . we report a case of pathologically proven simultaneous duodenal and colonic metastases about four years after nephrectomy for mixed clear and granular cell type renal cell carcinoma ( rcc ) . a 76-year - old female patient who had undergone a left radical nephrectomy 4 years previously for rcc presented with a 1-month history of dyspepsia and pain in the right upper abdomen . an abdominopelvic ct scan showed circumferential wall thickening with high enhancement at the second portion of the duodenum and additional enhancement of an irregular protruding mass into the lumen of the ascending colon . a gastroscopy showed a large and ulcerative protruding mass nearly obstructing the second portion of the duodenum . a colonoscopy revealed a polypoid , nodular and purplish mass in the ascending colon . microscopy of the biopsy specimen showed the features identical to those of the rcc which was resected 4 years earlier in this patient . we believe this to be the first case illustrating a metastatic renal cell carcinoma as simultaneous duodenal and colon masses .

sarcoidosis is a multisystem disease of undetermined cause which is characterized by the widespread presence of non - caseating granuloma . autopsy - based series report prevalence rates of renal involvement between 7 and 27% , and the disease is well known for its protean manifestations [ 24 ] . two non - mutually exclusive clinical and pathological patterns account for the great bulk of renal sarcoidosis , namely ( i ) nephrolithiasis and/or nephrocalcinosis - related to hypercalcaemia and hypercalciuria and ( ii ) interstitial nephritis with or without granulomatous formation [ 5 , 6 ] . in contrast , renal vascular involvement is believed to be an exceptional complication of sarcoidosis and extensive retrospective series have failed to document any such complication [ 5 , 6 ] . likewise , renal imaging findings are scanty and , if present , usually restricted to nephrocalcinosis , striated nephrograms or urinary tract obstruction [ 79 ] . here we report the case of a 22-year - old patient in whom the manifestation of renal sarcoidosis consisted of severe hypertension associated with bilateral vascular lesions and granulomatous interstitial nephritis . a 22-year - old white woman with a medical history of idiopathic epilepsy and sarcoidosis was referred to our institution for severe hypertension . six months before her referral to our institution she had been diagnosed with sarcoidosis during the follow - up of clinical features consistent with lfgren 's syndrome . during diagnostic work - up , blood pressure ( bp ) was shown to be within normal ranges ( 128/66 mmhg ) . corticosteroid therapy was considered unnecessary at that point and symptoms resolved within a couple of weeks . upon admission in our department , the patient 's bp was 169/123 mmhg under therapy of diltiazem 200 mg lp once daily , without clinical or echocardiographic repercussions . ( 68 mol / l , i.e. egfr = 98 ml / min/1.73 m , using the 4-variable mdrd [ modication of diet in renal disease ] ) formula . haematuria and urine infection were excluded by dipstick examination and microalbuminuria was within physiological ranges ( 8 mg / l ) . hormonal investigations , carried out under euvolaemic conditions , substantiated secondary hyperaldosteronism with elevated levels of plasma renin and aldosterone levels in the supine ( 75 mu / l and 31 ng / dl ; reference ranges 1025 and 2.814.4 , respectively ) and upright positions ( 298 mu / l ; and 2919 ng / dl ; reference ranges 1550 and 2001000 ) . the 24-h urine aldosterone levels were high ( 2487 ng/24 h ; reference range 5051694 ) . additional testing evidenced elevated levels of 25(oh ) vitamin d3 ( 85 nmol / l ; reference range 1157 ) but normal values of calcaemia ( 2.27 mmol / l ) , calcium urinary excretion ( 2.42 mmol / l ) and angiotensinogen - converting enzyme ( 11 mu / ml ) . an abdominal computed tomography scan ( ct ) revealed multiple bilateral wedge- and nodular - shaped hypodense foci affecting the renal cortex and medulla ( figure 1 ) , with no evidence of urinary tract obstruction . renal angiography was prompted by persistent suspicion of underlying renal ischaemia : multiple cortical and sub - cortical vascular defects were evidenced in both kidneys ( figure 2 ) . in view of the equivocal nature of the lesions , there were no signs of vessel destruction or of small - vessel angeitis in this specimen , and the rest of the examination was unremarkable ( figure 3 ) . 1.transverse contrast - enhanced abdominal ct during corticomedullary acquisition time displaying multiple , low - attenuation wedge - shaped foci seated in the cortex of both kidneys ( yellow arrows ) and low - attenuation corticomedullary nodules ( white arrowheads ) also present in both kidneys . 2.selective left kidney angiography showing multiple cortical and sub - cortical vascular defects ( black arrows ) . 3.first renal biopsy demonstrating the presence of sarcoidosic granulomatous inflammation in the vicinity of two normal interlobular arteries ( masson trichrome , original magnification 200 ) . transverse contrast - enhanced abdominal ct during corticomedullary acquisition time displaying multiple , low - attenuation wedge - shaped foci seated in the cortex of both kidneys ( yellow arrows ) and low - attenuation corticomedullary nodules ( white arrowheads ) also present in both kidneys . selective left kidney angiography showing multiple cortical and sub - cortical vascular defects ( black arrows ) . first renal biopsy demonstrating the presence of sarcoidosic granulomatous inflammation in the vicinity of two normal interlobular arteries ( masson trichrome , original magnification 200 ) . the patient was subsequently started on tapering doses of prednisone ( 1 mg / kg / day ) and ramipril ( 5 mg once daily ) . bp control was achieved and scr remained stable ( scr , 68 mol / l ; egfr , 98 ml / min/1.73 m ) . an abdominal mri performed 9 months after the initiation of corticosteroid therapy exhibited only minimal cortical defects in both kidneys whereas the lesions located in the medulla had receded . two years following the patient 's initial referral and progressive corticosteroid tapering , the patient presented with a recurrent bout of uveitis . concurrently , renal impairment was evidenced ( scr , 126 mol / l ; egfr , 45 ml / min/1.73 m ) . corticosteroids were reinitiated but proved to be unsuccessful as the patient 's kidney function failed to improve during the following year . three years after initial referral , ct imaging ( figure 4 ) substantiated major bilateral cortical atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted . there were also multiple left lateral aortic and caval lymph nodes , of increased size compared with the reference ct . 4.transverse contrast - enhanced abdominal ct during corticomedullary acquisition showing major bilateral kidney atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted ( white arrowheads ) . note the presence of multiple left lateral aortic and caval lymph nodes ( yellow arrows ) . transverse contrast - enhanced abdominal ct during corticomedullary acquisition showing major bilateral kidney atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted ( white arrowheads ) . note the presence of multiple left lateral aortic and caval lymph nodes ( yellow arrows ) . a 22-year - old white woman with a medical history of idiopathic epilepsy and sarcoidosis was referred to our institution for severe hypertension . six months before her referral to our institution she had been diagnosed with sarcoidosis during the follow - up of clinical features consistent with lfgren 's syndrome . during diagnostic work - up , blood pressure ( bp ) was shown to be within normal ranges ( 128/66 mmhg ) . corticosteroid therapy was considered unnecessary at that point and symptoms resolved within a couple of weeks . upon admission in our department , the patient 's bp was 169/123 mmhg under therapy of diltiazem 200 mg lp once daily , without clinical or echocardiographic repercussions . ( 68 mol / l , i.e. egfr = 98 ml / min/1.73 m , using the 4-variable mdrd [ modication of diet in renal disease ] ) formula . haematuria and urine infection were excluded by dipstick examination and microalbuminuria was within physiological ranges ( 8 mg / l ) . hormonal investigations , carried out under euvolaemic conditions , substantiated secondary hyperaldosteronism with elevated levels of plasma renin and aldosterone levels in the supine ( 75 mu / l and 31 ng / dl ; reference ranges 1025 and 2.814.4 , respectively ) and upright positions ( 298 mu / l ; and 2919 ng / dl ; reference ranges 1550 and 2001000 ) . the 24-h urine aldosterone levels were high ( 2487 ng/24 h ; reference range 5051694 ) . additional testing evidenced elevated levels of 25(oh ) vitamin d3 ( 85 nmol / l ; reference range 1157 ) but normal values of calcaemia ( 2.27 mmol / l ) , calcium urinary excretion ( 2.42 mmol / l ) and angiotensinogen - converting enzyme ( 11 mu / ml ) . an abdominal computed tomography scan ( ct ) revealed multiple bilateral wedge- and nodular - shaped hypodense foci affecting the renal cortex and medulla ( figure 1 ) , with no evidence of urinary tract obstruction . renal angiography was prompted by persistent suspicion of underlying renal ischaemia : multiple cortical and sub - cortical vascular defects were evidenced in both kidneys ( figure 2 ) . in view of the equivocal nature of the lesions , there were no signs of vessel destruction or of small - vessel angeitis in this specimen , and the rest of the examination was unremarkable ( figure 3 ) . 1.transverse contrast - enhanced abdominal ct during corticomedullary acquisition time displaying multiple , low - attenuation wedge - shaped foci seated in the cortex of both kidneys ( yellow arrows ) and low - attenuation corticomedullary nodules ( white arrowheads ) also present in both kidneys . 2.selective left kidney angiography showing multiple cortical and sub - cortical vascular defects ( black arrows ) . 3.first renal biopsy demonstrating the presence of sarcoidosic granulomatous inflammation in the vicinity of two normal interlobular arteries ( masson trichrome , original magnification 200 ) . transverse contrast - enhanced abdominal ct during corticomedullary acquisition time displaying multiple , low - attenuation wedge - shaped foci seated in the cortex of both kidneys ( yellow arrows ) and low - attenuation corticomedullary nodules ( white arrowheads ) also present in both kidneys . selective left kidney angiography showing multiple cortical and sub - cortical vascular defects ( black arrows ) . first renal biopsy demonstrating the presence of sarcoidosic granulomatous inflammation in the vicinity of two normal interlobular arteries ( masson trichrome , original magnification 200 ) . the patient was subsequently started on tapering doses of prednisone ( 1 mg / kg / day ) and ramipril ( 5 mg once daily ) . bp control was achieved and scr remained stable ( scr , 68 mol / l ; egfr , 98 ml / min/1.73 m ) . an abdominal mri performed 9 months after the initiation of corticosteroid therapy exhibited only minimal cortical defects in both kidneys whereas the lesions located in the medulla had receded . two years following the patient 's initial referral and progressive corticosteroid tapering , the patient presented with a recurrent bout of uveitis . concurrently , renal impairment was evidenced ( scr , 126 mol / l ; egfr , 45 ml / min/1.73 m ) . corticosteroids were reinitiated but proved to be unsuccessful as the patient 's kidney function failed to improve during the following year . three years after initial referral , ct imaging ( figure 4 ) substantiated major bilateral cortical atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted . there were also multiple left lateral aortic and caval lymph nodes , of increased size compared with the reference ct . 4.transverse contrast - enhanced abdominal ct during corticomedullary acquisition showing major bilateral kidney atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted ( white arrowheads ) . note the presence of multiple left lateral aortic and caval lymph nodes ( yellow arrows ) . transverse contrast - enhanced abdominal ct during corticomedullary acquisition showing major bilateral kidney atrophy , predominant in the right kidney . both kidneys displayed irregular contours with focal areas of cortical thinning where vascular defects had previously been noted ( white arrowheads ) . note the presence of multiple left lateral aortic and caval lymph nodes ( yellow arrows ) . this case reports an unusual presentation of renal sarcoidosis with prominent clinical features consisting of severe hypertension with bilateral vascular injury . despite a history strongly suggestive of sarcoidosis , renal biopsy indeed , the presence of nodular corticomedullar - located hypodense foci entertained the possibility of a coexisting tumoural process . only sparse case reports have documented this rare pseudonodular presentation [ 913 ] . in the even fewer instances where a renal biopsy was conducted , this lesional pattern was almost invariably related to extensive sarcoid granulomatous inflammation as in our case [ 1113 ] . renal sarcoidosis is usually associated with marked renal impairment on presentation . nevertheless , in three cases of bilateral , pseudotumoral , renal sarcoidosis ( out of a total of four reports ) the initial kidney function was shown to be normal as in the case presented here [ 1315 ] . renal pathology examination was also motivated by the triangular - shaped lesions located in the cortex which could be interpreted as post - ischaemic or evidence of infectious insults . renal angiography ultimately confirmed the suspicion of renal ischaemia by demonstrating multiple vascular voids in the renal cortex and sub - cortex . although no pathological process involving the renal arterioles could be demonstrated on pathology , this may be ascribed to a sampling bias given the focal distribution of the renal perfusion defects . regardless of the underlying mechanism , this process caused severe hypertension associated with secondary hyperaldosteronism and , in fine , widespread glomerular ischaemia and tubulointerstitial fibrosis . although sarcoidosis is known to embrace a wide variety of clinical and pathological manifestations ( table 1 ) , this case is different from the other rarely reported clinical patterns consisting of severe hypertension associated with small and medium - vessel injury . aside from its rarity , sarcoidosis - induced vascular injury has been shown to involve all types of renal vessels . in most cases , renal vascular damage is but one of the multiple manifestations of multisystem vasculitis , a rare life - threatening complication associated with sarcoidosis . in the case of small and medium - sized vascular involvement , the prominent clinical features also include pulmonary angiitis , cerebral vasculitis and skin sarcoidosis consistent with leukocytoclastic vasculitis and granulomatous vasculitis [ 17 , 18 ] . large vessel damage may either present as renal artery stenosis with concurring severe hypertension or aortic and renal aneurisms placing patients at high risk of subsequent rupture [ 18 , 20 ] . in stark contrast , at no point of the disease process did we record any extra - renal vascular involvement . furthermore , vascular wall involvement with elastic lamina dissolution as exemplified by previous case reports was not observed on pathological examination in our case [ 17 , 21 ] . table 1.summary of clinical and pathological patterns of renal sarcoidosispattern and/or cause of renal injuryfrequencyrenal function on presentationprominent laboratory featurespathological findings on kidney biopsyabdominal imaging findings ( on ct scan)granulomatous or non - granumolatous tubulointersititial nephritis+++altered , severe impairment is frequentmild proteinuria , microhematuria , sterile leukocyturia,+/ renal tubular acidosis+/ fanconi syndrometubulo - interstial nephritis+/ non necrotizing granuloma with epithelioid - like giant cells+/ schaumann bodiesnormal + + striated nephrogram + /pseudotumoral renal masses ( rare)nephrocalcinosis / nephrolithiasis++altered , severe impairment is frequenthypercalcaemia , hypercalciuria , elevated levels of 1 - 25(oh)vitd3+/renal tubular acidosis+/ fanconi syndromeintratubular and peritubular aggregation of calciumintrarenal calcifications+/ urinary obstructionglomerular diseaseraregenerally normal , mild to moderate impairment is possiblenephrotic range proteinuriamembranous nephritis + + + iga nephritis + + nrrenal vascular involvementvery rare??coexisting gin granuloma - associated vascular injuryrenal artery stenosis or aneurysmsmall- and medium - vessel occlusionmultisystem vascular involvementretroperitoneal fibrosisvery rare ? ? , no consistent data given the small number of cases ; a , most frequently described glomerular patterns ; gin , granulomatous interstitial nephritis ; nr , not relevant . , no consistent data given the small number of cases ; a , most frequently described glomerular patterns ; gin , granulomatous interstitial nephritis ; nr , not relevant . corticosteroids are regarded as the mainstay of renal sarcoidosis and retrospective studies have highlighted their effectiveness . however , several pitfalls should be kept in mind : ( i ) reversal of renal impairment is not universal and some patients may require other immunosuppressive therapy such as azathioprine and mycophenolate mofetil , ( ii ) residual renal failure is common , and not all patients regain their baseline renal function , ( iii ) relapses are frequent on cortiscosteroid weaning although patients may respond on reinitiation of therapy and ( iv ) response to therapy is negatively correlated to the extent of renal fibrosis [ 6 , 22 ] . however , by the time repeat imaging and biopsy were carried out the bilateral hypodense lesions thought to indicate the presence of granulomatous inflammation had resolved . instead , these lesions had given way to extensive renal fibrosis and the reintroduction of corticosteroids was of no avail . our case conveys further evidence that hypodense renal masses upon ct imaging may be regarded as a possible , albeit rare , manifestation of sarcoidosis . it also suggests that renal ischaemia and hypertension may arise via extrinsic compression from granulomatous inflammation in the absence of renal or extra - renal vasculitis . the occurrence of severe hypertension in a patient with a history of sarcoidosis should urge physicians to consider this complication . a.h . and d.n . provided the expertise in the imaging and histopathological examinations , respectively . c.r . undertook the data collection and analysis as well as the initial drafting of the report . although the basic principles of resuscitation were described by versalius more than 500 years ago , the practice of cardiopulmonary resuscitation in its modern form only starts 50 years ago . despite advances in the understanding and practices of airway management , ventilatory support , external cardiac compression and drug therapy the outcome of patients undergoing cardiopulmonary resuscitation remained poor . patients may have spontaneous circulation restored and admitted to the intensive care unit , but then developed complications related to ischaemic insult to the brain as well as to the rest of the body . the term post - resuscitation disease was coined by the russian resuscitologist vladimir a. negovsky in 1972 to describe the constellation of pathological processes caused by ischaemia and reperfusion associated with cardiac arrest and the subsequent resuscitation . this is more recently renamed post - cardiac arrest syndrome because " the term resuscitation is now used more broadly to include treatment of various shock states in which circulation has not ceased ... (and ) the term postresuscitation implies that the act of resuscitation has ended ... " 4 key components contribute to the development of this syndrome : 1 ) post - cardiac arrest brain injury ; 2 ) post - cardiac arrest myocardial dysfunction ; 3 ) systemic ischaemia / reperfusion response ; and 4 ) persistent precipitating pathology . there is evidence to support that proper management in the post - resuscitation phase can improve outcome of these patients and therapeutic hypothermia is one important component of such management . to avoid confusion , there is a need to define terminology used in relation to manipulation of body temperature : hypothermia is defined as core body temperature of less then 36 regardless of the cause.induced hypothermia is defined as an intentional reduction of a patient 's core temperature below 36.therapeutic hypothermia is defined as controlled induced hypothermia ; i.e. induced hypothermia with the potentially deleterious effects such a shivering , being controlled or suppressed.controlled or therapeutic normothermia is defined as bringing down core temperature in a patient with fever , and maintaining temperature within a range of 36 - 37.5 , with the potentially deleterious effects such a shivering , being controlled or suppressed . hypothermia is defined as core body temperature of less then 36 regardless of the cause . induced hypothermia is defined as an intentional reduction of a patient 's core temperature below 36. therapeutic hypothermia is defined as controlled induced hypothermia ; i.e. induced hypothermia with the potentially deleterious effects such a shivering , being controlled or suppressed . controlled or therapeutic normothermia is defined as bringing down core temperature in a patient with fever , and maintaining temperature within a range of 36 - 37.5 , with the potentially deleterious effects such a shivering , being controlled or suppressed . the degree of therapeutic hypothermia can mild ( 34.0 - 35.9 ) , moderate ( 32.0 - 33.9 ) , moderately deep ( 30.0 - 31.9 ) or deep ( early studies showed that induced hypothermia improved outcome in cardiac arrest patients . although both studies were small ( 12 patients had induced hypothermia in each study ) case series . however , further progress was hindered by 1 ) lack of understanding of the underlying pathophysiology , 2 ) lack of reliable methods to control body temperature , and 3 ) lack of intensive care facilities to manage possible complications . the early hypothesis that beneficial effect of hypothermia was due to reduction of metabolism leads to the adoption of more profound cooling ( around 30 ) . the result was serious complications which can not be handled effectively without the support of intensive care , which was then only at its infancy . further studies in the recent decades showed that the mechanism of cerebral protection by hypothermia is more complicated and neurological outcome can be improved by mild to moderate degree of hypothermia . two studies published in 2002 provided the evidence basis that hypothermia can improve neurological outcome . coupled with advances in technology in hypothermia and intensive care in general , the study population of both the study by bernard et al . and the haca study group are patients suffering from out - of - hospital ventricular fibrillations . there is evidence that cooling patients with presenting rhythms other than vf or vt following out - of - hospital arrests does no harm : 2 retrospective cohort studies found that the outcomes for patients presenting with pea / systole did not differ from those observed among the group presenting with vf or vt . there is little published evidence to support the use of therapeutic hypothermia following in - hospital cardiac arrest . an analysis of patients entered into arrich and erchacars group found that 13% of procedures were performed post in - hospital cardiac arrest . 43% of these were cooled and although the initiation of hypothermia was faster than that for the out - of- hospital arrests , there was no difference in outcome between the patients treated with hypothermia and those with normothermia . the haca in - hospital multi - center trial is currently investigating whether therapeutic hypothermia is beneficial for patients following in - hospital cardiac arrest . in both the haca and bernard studies , patients who are hypotensive ( map < 60 mmhg or sbp < 90 mmhg despite adrenaline infusion ) were excluded . the underlying reason being the possible deleterious effect of hypothermia on cardiovascular function and coagulation . however , there are several publications suggesting that post - cardiac arrest patients in cardiogenic shock could actually also benefit from moderate therapeutic hypothermia . reduction in platelet count and platelet dysfunction is noted when body temperature is dropped to < 35 ; and when temperature is below 33 , synthesis and kinetics of clotting enzymes and plasminogen activator inhibitors are also affected . however , it was found in a recent study which assessed the risk and severity of bleeding during simultaneous use of mild hypothermia and thrombolysis that bleeding risks were similar to historical controls treated with thrombolytics alone , although there was a trend toward more red blood cell units being required to reach target hematocrit in hypothermic patients who developed bleeding complications and needed transfusions . . the procedures of therapeutic hypothermia can be divided into 4 phases : induction , maintenance , de - cooling ( or rewarming ) and normothermia . this is the initial phase during which the patient 's body temperature is lowered to the target of 32 - 34. continuous core temperature measurement should be started before induction of therapeutic hypothermia to ensure that the patient 's body temperature is within the target range and to avoid overshoot . this is preferably achieved by bladder , rectal , central venous , or oesophageal measurement . however , it should be noticed that bladder temperature may poorly reflect core temperature if the patient is oliguric , and other monitoring sites are preferred . this phase is associated with many possible complications most of which can be minimized by rapid cooling . rapid cooling can be achieved by rapid bolus administration of 30 to 40 ml / kg cold ( 4 ) isotonic resuscitation fluid over 1 hour . despite theoretical problems this method when core temperature drops to 32 , metabolic rate decreases to 50 - 65% of normal . the issue of how to interpret arterial blood gases in hypothermic patients ( alpha - stat versus ph - stat ) is a topic of some controversy and readers are referred to review articles for detailed discussion . hypothermia can lead to hypokalaemia , hypophosphataemia and hypomagnesaemia as a result of intracellular shift as well as urinary loss because of tubular dysfunction . it is particularly important to monitor and replace potassium in order to avoid arrhythmia . on the other hand , it is the result of increased fat metabolism , leading to an increase in the levels of glycerol , free fatty acids , ketonic acids , and lactate . hyperglycaemia due to reduction of insulin secretion and insulin resistance should be controlled by close monitoring and insulin therapy . the main sources of relevant data come from patients with hypothermia induced during cardiac surgery . cardiac output decreased along with the heart rate ; however , the hypothermia - induced decrease in metabolic rate usually equaled or exceeded the decrease in cardiac output , so that the balance between supply and demand remained constant or improved . however , the actual cardiac output is also affected by the volume status and effect of sedation . systolic function of myocardium may actually be improved during mild to moderate hypothermia but diastolic function is usually impaired . it is important to note that the effect of hypothermia on myocardial contractility is strongly dependent on heart rate . if the heart rate is allowed to decrease along with the temperature , myocardial contractility as measured by systolic function usually increases , although there may be a mild degree of diastolic dysfunction . however , if the heart rate is artificially increased through administration of chronotropic drugs or a pacing wire , myocardial contractility decreases significantly . this phenomenon has been demonstrated in animal studies and also in patients undergoing cardiothoracic surgery . thus , the effect of hypothermia on myocardial function strongly depends on whether the heart rate is allowed to decrease [ 20 - 22 ] . maintaining euvolemia is one important aspect of management during therapeutic hypothermia and one challenge is cold induced diuresis . the underlying mechanisms leading to cold - induced diuresis include increased venous return caused by constriction of peripheral vessels ( particularly in the skin ) due to hypothermia - induced increases in plasma noradrenaline levels and activation of the sympathetic nerve system , activation of atrial natriuretic peptide , decreased levels of antidiuretic hormone and renal antidiuretic hormone receptor levels , and tubular dysfunction . if uncorrected , diuresis can cause hypotension as well as electrolyte depletion and increase in blood viscosity . the risk for hypovolemia increases significantly if the patient is simultaneously treated with diuretic agents such as mannitol . however , hypotension can be quite easily prevented by avoiding or promptly correcting hypovolemia , and by avoiding excessive stimulation of heart rate . while profound hypothermia of < 28 is associated with severe arrhythmia , mild to moderate hypothermia could stabilize membranes . study in normal human showed that the threshold for vasoconstriction is about 36.5 and that for shivering is 35.5. the threshold is slightly higher in female but the difference is < 0.5 . shivering of postoperative patients may lead to increased risk of morbid cardiac events especially in older patients with heart disease : the result of increased rate of metabolism and oxygen consumption , leading to excess work of breathing and tachycardia . however , the situation may be different in sedated patients . while hypothermia in an awake patient causes tachycardia , inducing hypothermia intentionally in sedated patients shivering will increase oxygen consumption , but as the patient is on mechanical ventilation , there will be no increase in the work of breathing . nevertheless , it is important to prevent or aggressively treat shivering because it significantly complicates hypothermia induction , and leads to an undesirable increase in metabolic rate and oxygen consumption . shivering may be controlled by one and more of the following methods : 1 ) drugs lowering shivering threshold , e.g. acetaminophen ( paracetamol ) , aspirin , and nonsteroidal antiinflammatory drugs ; 2 ) drugs suppressing shivering response paralyzing agents , sedatives , opiates , and others ; and/or 3 ) skin counterwarming . drugs lowering shivering threshold are not quite effective in general . of the drugs which suppress shivering , it was found that there is significant variation in sedation protocols amongst the 68 icus included in the selected studies . midazolam ( 5 mg / h to 0.3 mg / kg / h ) being most commonly used , followed by propofol ( up to 6 mg / kg / h ) . a quarter of icus do not use any analgesic , the rest use either fentanyl ( 0.5 - 10 mcg / kg / h ) or morphine . use of neuromuscular blocking agents , however , is associated with several disadvantages some of which can be reduced by adequate sedation : 1 ) attempts of the brain to generate a shivering response will not cease ; 2 ) they can mask seizure ; 3 ) they lack of vasodilating effect of sedatives ; and 4 ) their use is associated with development of critical illness polyneuromyopathy . the importance of adequate sedation and suppression of hypothermia - induced stress responses is underscored by observations from animal experiments suggesting that some or all of hypothermia 's neuroprotective effects can be lost if cooling is used in non - sedated animals . it was found that 4 increase in skin temperature could " compensate " for 1 drop in core temperature to prevent a shivering response . some areas of the body ( hands , feet and face ) have a higher concentration of " temperature sensors " and warming of those areas could have a greater effect on suppressing shivering . by reducing shivering , counterwarming can reduce metabolic rate and oxygen consumption of hypothermic patients . during this phase , the goal is to maintain core temperature at 32 - 34 for 12 - 24 hours . the patient is usually more stable during this phase with fewer disturbances in their haemodynamic , volume or electrolyte status , as well as less shivering . however , the patient is subjected to other hazards including changes in pharmacokinetics , nosocomial infections and bedsores . conventionally , patients are maintained cool by surface cooling through exposure , placing ice packs to the neck , groins and axillae , and/or spraying with water . rubber cooling blankets can also be used but should be placed over the patient . placing those blankets under the patient can lead to skin damage due to vasoconstriction in already pressurized areas . although widely available and inexpensive , conventional surface cooling is difficult to control as there is no internal feedback mechanism . these may be classified as non - invasive surface cooling devices such as hydrogel - coated water circulating pads or water - circulating wrapping garments , and invasive core cooling devices using intravascular catheters . a problem with surface cooling is that 40 - 90% the patient 's surface area needs to be covered and carries a risk of skin lesions . but they can be started immediately using nurse - driven protocols without direct physician intervention . core - cooling methods , on the other hand , are highly reliable once the catheter is in place , with a cooling rate of 2.0 - 4.5/hour . however , insertion of catheter is required and it can lead to complications such as thrombosis and infection . even short duration of hypothermia during perioperative period for intracranial aneurysm increase the risk of postoperative bacteraemia . a study has shown that 3 days of prophylaxis with ampicillin - sulbactam can reduce early onset nosocomial pneumonia but not the incidence of late - onset nosocomial pneumonia . after 12 - 24 hours of cooling , patient can be allowed to warm up slowly . the goal should be 0.2 - 0.33 per hour until the patient is 36.5 - 37.0. more rapid warming must be prevented to avoid electrolyte disturbance due to transcellular shift , hypoglycaemia due to increased insulin sensitivity , as well as aggravated brain destruction . this can be prevented by slow rewarming , allowing the kidneys to excrete the excess potassium . however , this should not be a reason to accept hypokalaemia in the induction or maintenance phase . for patients with renal dysfunction , renal replacement therapy this may be the result of nosocomial infection or part of the post - cardiac arrest syndrome . furthermore , cerebrovascular reactivity may be impaired following hypothermia treatment and thus increase the potential harmful effects of fever even more . there was a case report in which a post - cardiac arrest patient who initially improved after rewarming deteriorated after development of fever and subsequently died . it is recommended that patients should be maintained normothermic after decooling until 72 hours have elapsed since restoration of spontaneous circulation . current evidence supports that induction of therapeutic hypothermia in selected patients after cardiac arrest can improve neurological outcome . lack of familiarity and lack of a concrete protocol are the most important amongst the list of perceived barriers . it is hoped that by summarizing the current state of knowledge on the subject and highlighting issues on clinical management will enable more patients to benefit from the therapy .

renal sarcoidosis embraces a wide variety of clinical patterns . renal vascular involvement has seldom been reported and usually in the setting of systemic vasculitis . we report the case of a 22-year - old patient in whom inaugural manifestation of renal sarcoidosis consisted of severe hypertension associated with bilateral perfusion defects and tumour - like nodules . in the setting of renal sarcoidosis , our case suggests that renin - dependant hypertension may arise from renal ischaemia as a result of extrinsic compression of kidney blood vessels due to severe granulomatous inflammation . pioneer works on therapeutic hypothermia ( th ) half a century ago already showed promising results but clinical application was limited by a lack of understanding of the underlying pathophysiology , lack of reliable method for temperature control and lack of intensive care facilities to deal with possible complications . more recently , 2 studies in 2002 supported the application of moderate th ( 32.0 - 34.0 ) in post - cardiac arrest patients . although the studies included only patients suffering from out - of - hospital vf , many icus world - wide are applying the therapy to all post - cardiac arrest patients irrespective of site or presenting rhythm . while primary coagulopathy and cardiogenic shock are usually stated as relative contraindications , evidences are accumulating to support the application of th in patients with cardiogenic shock . th can be divided into 4 phases : induction , maintenance , de - cooling and normothermia . induction is usually achieved by infusion of cold isotonic fluid . the precautions included avoidance of over - cooling , hypokalaemia , hyperglycaemia , and shivering . th can be maintained by many different methods , varying in their level of invasiveness , cost and effectiveness . issues including changes in pharmacokinetics and haemodynamics , and susceptibility to infection need to the addressed . the optimal duration of maintenance is unknown but the usual practice is 12 - 24 hours . de - cooling and rewarming is especially challenging because complications can be serious if temperature rise by more than 1 every 3 - 5 hours . life - theatening hyperkalaemia can occur especially if patient suffers from renal insufficiency . fever is a frequent complication either due to infection or post - cardiac arrest syndrome but patient must be kept normothermic for 72 hours .

myctophids ( myctophidae ) are mesopelagic fishes distributed in all the world s oceans ( dalpadado and gjster 1988 ; cherel et al . 2010 ) , playing an important role in the marine ecosystems ( gjster and kawaguchi 1980 ; cherel et al . 2010 ) as trophic links between zooplankton ( kinzer and schulz 1985 ; shreeve et al . 2009 ) and piscivorous predators ( giske et al . 1990 ; markaida and sosa - nishizaki 2003 ; dokster et al . they are also likely to play a major role in the vertical flux of organic matter from the upper productive layer to deeper layers ( radchenko 2007 ; hernandez - leon et al . their vertical distribution and diel vertical migration behavior affect such interactions and processes , making it important to reveal these patterns . the myctophid glacier lanternfish ( benthosema glaciale ) and the sternoptychidae pearlside ( maurolicus muelleri ) are the dominant mesopelagic fish in norwegian fjords ( aksnes et al . 2004 ; kristoffersen and salvanes 2009 ) . in masfjorden , pearlside dominates acoustic scattering layers ( sl s ) shallower than 200 m , while glacier lanternfish dominates the sl s below 200250 m ( giske et al . 1990 ; bagien et al . 2001 ; kaartvedt et al . 2009 ; dypvik et al . diel vertical migration ( dvm ) is commonly referred to as a trade - off between feeding opportunities and predation risk induced by changes in light intensity ( clark and levy 1988 ; pearre 2003 ; cohen and forward 2009 ; ringelberg 2010 ) . thus , light is the proximate cause of dvm influencing the time of migration and vertical extent of migration ( ringelberg and van gool 2003 ; staby and aksnes 2011 ) , while the distribution of food and predation risk are regarded as major drivers for fish vertical distribution and migration ( clark and levy 1988 ; bailey 1989 ; neilson and perry 1990 ; sutton and hopkins 1996 ) . however , size ( busch and mehner 2012 ) , internal factors such as energy reserves ( hays et al . 2001 ) and hunger ( pearre 2003 ) , and external factors such as currents ( bennett et al . 2002 ) and temperature ( wurtsbaugh and neverman 1988 ; sogard and olla 1996 ; mehner et al . 2010 ) may also influence vertical distribution and migration . in normal dvm ( ndvm ) , individuals ascend toward the surface at night before descending to deeper waters during the day . in the less common inverse dvm ( idvm ) , individuals ascend in the water column at daytime and descend during the night ( pearre 2003 ) . glacier lanternfish is known to carry out ndvm ( halliday 1970 ; roe and badcock 1984 ; sameoto 1988 ) , idvm ( kaartvedt et al . 2012 ) or display no diel vertical migration ( nodvm ) ( roe and badcock 1984 ; albikovskaya 1988 ; kaartvedt et al . the depth distribution of myctophids is size dependent with larger fish distributed deeper than smaller individuals ( willis and pearcy 1980 ; gartner et al . 1994 ) , vision ( capabilities ) for detecting prey ( warrant and locket 2004 ) , internal state ( satiation and hunger ) ( cailliet and ebeling 1990 ; staby et al . 2011 ) , and motivation ( rosland and giske 1997 ; busch and mehner 2012 ) are expected to result in a mixture of migration patterns , which may occur simultaneously . glacier lanternfish feeds on a variety of zooplankton ( gjster 1973 ; roe and badcock 1984 ; sameoto 1988 ) , but seems to prefer calanoid copepods , especially calanus ( sameoto 1988 , 1989 ; balio and aksnes 1993 ; dypvik et al . 2012 ) . the main pattern in seasonal zooplankton abundance and vertical distribution at the site for this study is established from previous studies at the same or adjacent locations . in spring and summer , most zooplankton are distributed in the upper part ( < 30 m ) of the water column ( rasmussen and giske 1994 ) . however , during autumn and winter , the highest biomass of zooplankton is below 150 m ( giske et al . this is because zooplankton vanishes from upper layers as primary production declines , but also because of seasonal vertical migration among the main calanoid copepod species , calanus spp . , which leaves upper waters , descending for overwintering in mid - waters ( bagien et al . myctophids are capable of feeding at mesopelagic depths ( roe and badcock 1984 ; sameoto 1988 ; pusch et al . 2004 ) , so that the seasonal migration of calanus to deep waters may represent an important food source in the daytime habitat of the fish ( dypvik et al . in norwegian fjords , glacier lanternfish exercise a strong predation pressure on overwintering calanus ( bagien et al . mesopelagic fish can be studied by use of echo sounders as they tend to aggregate into sl s ( holton 1969 ; god et al . normally , acoustic studies of mesopelagic fish are carried out in periods restricted by time or seasonality ( collins et al . 2009 ) , and to our knowledge , there is no systematic study addressing how migration patterns in a population of glacier lanternfish , or other myctophids , may vary throughout a year . however , the use of moored echo sounders can give long - time acoustic data series ( brierley et al . here we take advantage of the unique opportunity for a long - term study offered by a deep fjord where populations of mesopelagic fish are easily accessible . we present data from 16 months of continuous acoustic registrations ( july 2007october 2008 ) , enabling us to address the seasonal patterns of diel vertical behavior , unveiling the relative occurrence and consistency of ndvm , idvm , and nodvm by glacier lanternfish . at the outset of this study , we hypothesized that the patterns of glacier lanternfish dvm would vary seasonally , as recently documented for the pearlside ( staby et al . given the general seasonal zooplankton dynamics of norwegian fjords , we hypothesize that during spring and summer , when the abundance of potential prey is high in near - surface waters , the glacier lanternfish migrate to the upper part of the water column at night ( ndvm ) to feed , avoiding visual predators in the bright surface waters during daytime . conversely , we hypothesize that during autumn and winter , when food is sparse in upper waters , and at seasonal high in mid - waters , as seasonally migrating copepods have descended to their overwintering habitat , glacier lanternfish restrain from migrations to the surface at night , rather exploiting the prey in mid - waters by performing idvm toward favored light conditions in shallower waters during daytime ( dypvik et al . furthermore , for the nodvm layer , we investigate two alternative hypotheses : ( 1 ) the population really does split into migrating and non - migrating components ( pearcy et al . 1979 ) or ( 2 ) migrations are undertaken also among the nodvm component , but not detected by standard methods because they are asynchronous , so that a proportion of the population always is present in deep waters ( sutton and hopkins 1996 ; pearre 2003 ) . since our approach facilitates studies of individuals , we have the possibility of addressing these competing hypotheses . the study was undertaken in masfjorden ( ~6052n , ~524e ) , norway ( fig . 1 ) . the deeper parts of the water column ( ~200390 m ) are particularly in focus as glacier lanternfish dominates the acoustic backscattering , and idvm and nodvm occur in this interval ( dypvik et al . depicting location of the bottom - mounted echo sounder ( gray circle ) at 392 m depth in masfjorden , norway map depicting location of the bottom - mounted echo sounder ( gray circle ) at 392 m depth in masfjorden , norway a mooring with an upward - facing 38 khz simrad ek60 split - beam echo sounder ( 7.1 beam width ) , cabled to shore for power and transmission of data , was deployed at 392 m in july 2007 and retrieved in october 2008 . detailed descriptions of the echo sounder system and parameter settings are given in kaartvedt et al . data were recorded continuously , apart for intermittent periods following power failures , giving a total of 218 full days of records spanning all seasons . a minimum number of 4 days per month were included in this study ( i.e. sept . 2007 and we classify september november as autumn , december february as winter , march may as spring , and june august as summer . the results are presented as average echograms illustrating the average sv ( db ) values for the entire water column ( ~0390 m ) for each month , and in greater detail from selected months representing each season for the section ~200390 m , which is of particular interest for this study ( called monthly echograms ) . sv values are logarithmic measurements of the volume acoustic backscattering or accumulated ts ( target strength ) , ts representing the echo of a single individual ( maclennan and simmonds 1992 ) . the seasonal patterns of the vertical distribution and migration of glacier lanternfish is assessed by studying the scattering layers in monthly echograms . these monthly echograms were made in matlab by dividing each day into 30 s intervals , and for each time interval average all the pings for the given month . in this way , all the acoustic records from the entire registration period are included in the presentations . in addition , to better illustrate any activity in the nodvm layer , which by definition was expected to have little vertical migration , we randomly picked 1 day for each season ( 18 october 2007 ; 2 february ; 12 march 2008 ; 8 august 2008 ) , focusing on the 300390 m depth interval . for these days , we furthermore selectively chose examples where vertical swimming was seen in the compressed , daily echograms , and portrayed these at higher resolution for better depicting individual behavior . abundance estimates to identify the variation in concentration of glacier lanternfish in different depth intervals between day and night were made by echo integration . this was done with day and depth divided into blocks of 1 h and 10 m respectively between ~250 and 390 m for days where 24 h of echo data were successfully retrieved ( from min . time for sunrise and sunset was set to the 15th of each corresponding month , as an approximate for each month , determining time allocated to day and night . glacier lanternfish makes up the main part of the volume backscattering ( sv ) at 90 db , while larger fish results in sv values greater than 64 db ( bagien et al . 2001 ) . thus , values retrieved by echo integration at 64 db were subtracted from the echo integration at 90 db to exclude fish larger than glacier lanternfish ( bagien et al . the concentrations ( individuals m ) were derived from measurements of sv and target strength ( ts , see description below ) by dividing the linear sv values with the linear ts ( maclennan and simmonds 1992 ) . ts depends on the size , anatomy , and behavior of the organism , as well as the frequency of the echo sounder ( maclennan and simmonds 1992 ) . for precise estimation of concentration , the ts of glacier lanternfish was obtained monthly by means of automatic target tracking ( tt ) , performing tt for 3 days of every month within a range of 1050 m from the transducer ( ~340380 m depth ) . this limited range was chosen as the resolution of acoustic data decreases with distance from the transducer , and so does the precision of the ts estimates . minimum track length was set at 10 ping , maximum ping gap to 1 ping and gating range to 0.3 m during tracking . ts values between 65 and 50 db were regarded as glacier lanternfish , as ts values stronger than 50 db indicate fish of greater size ( foote 1980 ; maclennan and simmonds 1992 ) . a minimum value of 65 db was chosen in accordance with a previous acoustic study of glacier lanternfish ( torgersen and kaartvedt 2001 ) . tracks of individual fish obtained by tt were also used for analysis of swimming behavior . vertical swimming speeds of individuals were analyzed together with visual inspection of daily 24 h echograms in order to investigate any asynchronicity in the dvm pattern of the deeper living part of the population . both tt and echo integration were performed in the post processing program sonar_5 pro version 5.9.9 ( balk and lindem 2007 ) . the migration patterns below 250 m as assessed from the monthly echograms were compared with changes in concentration as assessed based on combining the measurements of sv and ts . the maximum concentration of glacier lanternfish estimated below 250 m each day was assumed to reflect the total population below 250 m. increase and/or decrease in concentration , before and after the time of migration of a specific layer of glacier lanternfish , was used as a proxy for the relative proportion of glacier lanternfish with nodvm , idvm , and ndvm . the speed of migration by individuals in sl s was calculated by analyzing the distance of descent / ascent over time in sl s . the continuous acoustic measurements were complemented by sampling during research cruises 14 november 2007 with r / v haakon mosby ( university of bergen , imr ) and 37 october 2008 with r / v trygve braarud ( university of oslo ) . results from the physical oceanography , zooplankton , and trawling studies from these field campaigns are used in the interpretations , but are presented elsewhere ( staby et al . the behavioral patterns described in this study can with confidence be ascribed to glacier lanternfish . previous studies covering spring and summer ( rasmussen and giske 1994 ; kaartvedt et al . unpublished ) , autumn ( bagien et al . 2001 ; kaartvedt et al . 2009 ) , winter ( giske et al . 1990 ; balio and aksnes 1993 ; bagien et al . 2001 ) , and trawl data obtained during the present study ( staby et al . 2011 ; dypvik et al . 2012 ) , as well as subsequent studies ( kaartvedt et al . unpublished ) have shown that glacier lanternfish are the main cause of backscattering deeper than 250 m in masfjorden . catches at these depths consist of specimens > 4.5 cm , with a prevalence of individuals > 6 cm below 300 m ( kaartvedt et al . 2009 ; dypvik et al . 2012 ) , that is adult individuals ( gjster 1981 ) . pelagic shrimps are also common in the deep waters of masfjorden ( kaartvedt et al . 1988 , 2009 ) and may contribute somewhat to the backscatter . however , the sl s below 200 m in masfjorden are pronounced also at 18 khz ( kaartvedt et al . 2008 ) , signifying that the backscatter mainly consists of mesopelagic fish ( torgersen and kaartvedt 2001 ; love et al . 2004 ; god et al . 2009 ; kloser et al . 2009 ) . furthermore , the average ts of the targets studied here ( see results ; target strength and concentration estimates in deep water ) is in accordance with previous studies of lanternfishes ( torgersen and kaartvedt 2001 ; yasuma et al . 2009 ) , while shrimps have a ts about one magnitude weaker than that of glacier lanternfish ( benoit - bird and au 2001 ; torgersen and kaartvedt 2001 ) . in summer , there were four sl s performing ndvm to the surface at night , and one deep layer with no apparent diel behavior ( fig . 2 ) . the upper of these layers ( ~70200 m ) were ascribed to pearlside as they are found to dominate at these depths ( the seasonal dvm patterns of pearlside are discussed in staby et al . the two deeper layers ( > 250 m during daytime ) were ascribed to glacier lanternfish with ndvm and nodvm , respectively . zooming in on the deeper parts of the water column illustrates more clearly how organisms carrying out ndvm in summer descended to waters below ~250 m at daytime , and ascended at night ( fig . 2monthly average echograms from the 38-khz echo sounder covering the water column ( ~0390 m ) for 24 h in july 2007october 2008 . the dominant signal ( green ) and backscattering at shallower depth ( < 200 m ) is dominated by pearlside , while glacier lanternfish dominate below ~250 the boxes indicate the intervals where the concentration ( individuals m ) of glacier lanternfish is calculated ( see fig . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . the s v - thresholds are set to 50 and 85 db for the best possible illustration of the sl s . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . 3monthly average echograms from the 38-khz echo sounder ( covering 24 h ) illustrating the deeper part of the water column ( ~200390 m ) in summer ( represented by august 2008 ) , autumn ( represented by september 2007 ) , winter ( represented by january 2008 ) , and spring ( represented by may 2008 ) . arrows in echograms indicate the different behavioral patterns ; no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) monthly average echograms from the 38-khz echo sounder covering the water column ( ~0390 m ) for 24 h in july 2007october 2008 . the dominant signal ( green ) and backscattering at shallower depth ( < 200 m ) is dominated by pearlside , while glacier lanternfish dominate below ~250 the boxes indicate the intervals where the concentration ( individuals m ) of glacier lanternfish is calculated ( see fig . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . the s v - thresholds are set to 50 and 85 db for the best possible illustration of the sl s . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) monthly average echograms from the 38-khz echo sounder ( covering 24 h ) illustrating the deeper part of the water column ( ~200390 m ) in summer ( represented by august 2008 ) , autumn ( represented by september 2007 ) , winter ( represented by january 2008 ) , and spring ( represented by may 2008 ) . arrows in echograms indicate the different behavioral patterns ; no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) in autumn , ndvm was still present above ~200 m , but was less noticeable below ~200 m ( fig . 2 ) . idvm of acoustic targets ascribed to glacier lanternfish was now evident below the pearlside sl ( figs . 2 , 3 ) . an apparent non - migrating layer of glacier lanternfish occurred from 300 m to the bottom ( figs . 2 , 3 ) . in winter , there were strong echoes of vertical migrating juvenile pearlside , and largely non - migrating adult pearlside in the upper 200 m ( fig . 2 ) . the sl of individuals with idvm ( glacier lanternfish ) was still apparent , although less pronounced than in autumn ( figs . 2 , 3 ) . the deepest non - migrating layer was now at its seasonal high in level of backscatter ( fig . 2 ) . in spring , several sl s with ndvm appeared between ~100210 m in daytime ( march and april ) and in the upper ~100 m at night ( fig . 2 ) . , individuals performing ndvm descended to ~300 m in daytime , indicating a large portion of the glacier lanternfish population with this behavior ( figs . 2 , 3 ) . m ( glacier lanternfish ) , yet with a seasonal low in backscatter ( fig . 2 ) . estimated proportions with different dvm modes were calculated for each month based on the echograms and the echo integration from 250 to 390 m ( fig . ndvm was performed by ~2555 % of these fish in summer , while this behavior ceased in autumn and winter ( fig . however , individuals performing ndvm re - appeared in early spring , and the proportion performing ndvm increased toward late spring ( from ~13 % in march to ~51 % in may ) ( fig . 4 ) . the proportion performing idvm increased from late summer ( ~15 % in august ) to the beginning of autumn ( 70 % in september 2007 ) , then decreased throughout the winter ( 24 % in february 2008 ) . in spring and the first part of summer , idvm was not recorded ( fig . nodvm was suggested for ~4575 % of the population at these depths during spring and summer , but this proportion decreased from summer to autumn ( ~3040 % in september 2007 and 2008 ) , before it increased toward the winter months ( fig . 4the relative frequency of the population of glacier lanternfish ( found below 250 m during daytime ) with different dvm behaviors from july 2007october 2008 . no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) are illustrated by black , white , and gray colors , respectively the relative frequency of the population of glacier lanternfish ( found below 250 m during daytime ) with different dvm behaviors from july 2007october 2008 . no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) are illustrated by black , white , and gray colors , respectively visual inspection of 24 h echograms suggested little systematic vertical swimming among the nodvm component in relation to time of day , as exemplified by 1 day each season in fig . fish at these depths commonly appeared to drift up and down in a consistent pattern suggesting internal wave motions ( fig . the limited vertical movement was reflected in the results from the year - round tt . of more than 200,000 tracks retrieved by tt however , intermittent vertical swimming was recorded in which individuals ascended or descended in a step - wise pattern ( fig . the maximum vertical swimming speed detected was ~18.4 cm / s . in comparison , the approximately ascending and descending speeds of the migrating layers were 34 cm / s for the ndvm layer and 0.50.8 cm / s for the idvm layer . in addition to such marked vertical relocations , seemingly passively drifting fishes once in a while slightly adjusted their vertical distribution , subsequently taking up their apparent torpid behavior.fig . 5 a d 24-h echograms from the bottom - mounted 38 khz echo sounder on 8 august 2008 ( a ) , 18 october 2007 ( b ) , 2 february ( c ) , and 12 march 2008 ( d ) . the echograms are zoomed in at ~300390 m. the outlined boxes are presented in higher resolution in ( e h ) . the color scale refers to average volume backscattering ( s v ) ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset . time is given as utc ( local standard time 1 h ) a d 24-h echograms from the bottom - mounted 38 khz echo sounder on 8 august 2008 ( a ) , 18 october 2007 ( b ) , 2 february ( c ) , and 12 march 2008 ( d ) . the echograms are zoomed in at ~300390 m. the outlined boxes are presented in higher resolution in ( e h ) . the color scale refers to average volume backscattering ( s v ) ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset . time is given as utc ( local standard time 1 h ) the average ts ( db ) of glacier lanternfish as measured between ~340 and 380 m were always in the range of approximately 57.5 to 59.5 db . within this limited range , the ts distribution of glacier lanternfish showed two peaks ( in both summers ) and decreased from the autumn to spring ( fig . 6average ts ( db ) of glacier lanternfish ( total n = 209,768 ) calculated from ~340 to 380 m for each month throughout the study period ( july 2007october 2008 ) average ts ( db ) of glacier lanternfish ( total n = 209,768 ) calculated from ~340 to 380 m for each month throughout the study period ( july 2007october 2008 ) the ts values were used in assessing the numerical densities of glacier lanternfish . the concentration of fish was usually in the range of 0.0050.015 individuals m ( fig . however , the maximum density , just below 0.02 individuals m , was recorded in july 2008 in the deepest interval ( 300390 m ) , and in october 2008 in the shallower interval ( 250300 m ) ( fig . 7a , b).fig . 7 a , b estimated monthly average concentration ( individuals per m ) of glacier lanternfish during day and night from ( a ) 250300 m and ( b ) 300390 m during july 2007october 2008 . the white and black squares indicate the median daytime and nighttime concentration estimates , respectively . the black lines illustrate the 1st and 3rd quartile , while the x indicates the average estimated concentration a , b estimated monthly average concentration ( individuals per m ) of glacier lanternfish during day and night from ( a ) 250300 m and ( b ) 300390 m during july 2007october 2008 . the white and black squares indicate the median daytime and nighttime concentration estimates , respectively . the black lines illustrate the 1st and 3rd quartile , while the x indicates the average estimated concentration in spring and summer , when ndvm is the dominating mode of migration , the daytime concentration of the interval ~250300 m was higher than during night ( fig . below 300 m , where the nodvm mode were dominating , relatively little concentration fluctuations between day and night occurred , except for june august 2008 ( fig . 7b ) when the ndvm layers entered those depths at daytime ( fig . 2 ) . in the months where idvm were the dominating mode of migration ( i.e. september february ) , the concentration of glacier lanternfish decreased during daytime in the interval ~250300 m ( fig . 7a ) , as the inverse vertical migrators that were distributed from ~220 to 300 m during nighttime ascended to mid - waters ( in the lower part of the strong sl between ~150220 m ) during the day ( figs . 2 , 3 ) . in summer , there were four sl s performing ndvm to the surface at night , and one deep layer with no apparent diel behavior ( fig . 2 ) . the upper of these layers ( ~70200 m ) were ascribed to pearlside as they are found to dominate at these depths ( the seasonal dvm patterns of pearlside are discussed in staby et al . the two deeper layers ( > 250 m during daytime ) were ascribed to glacier lanternfish with ndvm and nodvm , respectively . zooming in on the deeper parts of the water column illustrates more clearly how organisms carrying out ndvm in summer descended to waters below ~250 m at daytime , and ascended at night ( fig . 2monthly average echograms from the 38-khz echo sounder covering the water column ( ~0390 m ) for 24 h in july 2007october 2008 . the dominant signal ( green ) and backscattering at shallower depth ( < 200 m ) is dominated by pearlside , while glacier lanternfish dominate below ~250 the boxes indicate the intervals where the concentration ( individuals m ) of glacier lanternfish is calculated ( see fig . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . the s v - thresholds are set to 50 and 85 db for the best possible illustration of the sl s . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . 3monthly average echograms from the 38-khz echo sounder ( covering 24 h ) illustrating the deeper part of the water column ( ~200390 m ) in summer ( represented by august 2008 ) , autumn ( represented by september 2007 ) , winter ( represented by january 2008 ) , and spring ( represented by may 2008 ) . arrows in echograms indicate the different behavioral patterns ; no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) monthly average echograms from the 38-khz echo sounder covering the water column ( ~0390 m ) for 24 h in july 2007october 2008 . the dominant signal ( green ) and backscattering at shallower depth ( < 200 m ) is dominated by pearlside , while glacier lanternfish dominate below ~250 the boxes indicate the intervals where the concentration ( individuals m ) of glacier lanternfish is calculated ( see fig . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . the s v - thresholds are set to 50 and 85 db for the best possible illustration of the sl s . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) monthly average echograms from the 38-khz echo sounder ( covering 24 h ) illustrating the deeper part of the water column ( ~200390 m ) in summer ( represented by august 2008 ) , autumn ( represented by september 2007 ) , winter ( represented by january 2008 ) , and spring ( represented by may 2008 ) . arrows in echograms indicate the different behavioral patterns ; no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) . the color scale refers to average volume backscattering ( s v ) for each month ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset for the 15th each month . time is given as utc ( local standard time 1 h ) in autumn , ndvm was still present above ~200 m , but was less noticeable below ~200 m ( fig . 2 ) . idvm of acoustic targets ascribed to glacier lanternfish was now evident below the pearlside sl ( figs . 2 , 3 ) . an apparent non - migrating layer of glacier lanternfish occurred from 300 m to the bottom ( figs . 2 , 3 ) . in winter , there were strong echoes of vertical migrating juvenile pearlside , and largely non - migrating adult pearlside in the upper 200 m ( fig . 2 ) . the sl of individuals with idvm ( glacier lanternfish ) was still apparent , although less pronounced than in autumn ( figs . 2 , 3 ) . the deepest non - migrating layer was now at its seasonal high in level of backscatter ( fig . 2 ) . in spring , several sl s with ndvm appeared between ~100210 m in daytime ( march and april ) and in the upper ~100 m at night ( fig . 2 ) . , individuals performing ndvm descended to ~300 m in daytime , indicating a large portion of the glacier lanternfish population with this behavior ( figs . 2 , 3 ) . m ( glacier lanternfish ) , yet with a seasonal low in backscatter ( fig . 2 ) . estimated proportions with different dvm modes were calculated for each month based on the echograms and the echo integration from 250 to 390 m ( fig . 4 ) . ndvm was performed by ~2555 % of these fish in summer , while this behavior ceased in autumn and winter ( fig . however , individuals performing ndvm re - appeared in early spring , and the proportion performing ndvm increased toward late spring ( from ~13 % in march to ~51 % in may ) ( fig . the proportion performing idvm increased from late summer ( ~15 % in august ) to the beginning of autumn ( 70 % in september 2007 ) , then decreased throughout the winter ( 24 % in february 2008 ) . in spring and the first part of summer , nodvm was suggested for ~4575 % of the population at these depths during spring and summer , but this proportion decreased from summer to autumn ( ~3040 % in september 2007 and 2008 ) , before it increased toward the winter months ( fig . . 4the relative frequency of the population of glacier lanternfish ( found below 250 m during daytime ) with different dvm behaviors from july 2007october 2008 . no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) are illustrated by black , white , and gray colors , respectively the relative frequency of the population of glacier lanternfish ( found below 250 m during daytime ) with different dvm behaviors from july 2007october 2008 . no diel vertical migration ( nodvm ) , inverse diel vertical migration ( idvm ) , and normal diel vertical migration ( ndvm ) are illustrated by black , white , and gray colors , respectively visual inspection of 24 h echograms suggested little systematic vertical swimming among the nodvm component in relation to time of day , as exemplified by 1 day each season in fig . fish at these depths commonly appeared to drift up and down in a consistent pattern suggesting internal wave motions ( fig . the limited vertical movement was reflected in the results from the year - round tt . of more than 200,000 tracks retrieved by tt , ~85 % revealed vertical relocation speed < 0.4 cm / s . however , intermittent vertical swimming was recorded in which individuals ascended or descended in a step - wise pattern ( fig . 5e h ) . the maximum vertical swimming speed detected was ~18.4 cm / s . in comparison , the approximately ascending and descending speeds of the migrating layers were 34 cm / s for the ndvm layer and 0.50.8 cm / s for the idvm layer . in addition to such marked vertical relocations , seemingly passively drifting fishes once in a while slightly adjusted their vertical distribution , subsequently taking up their apparent torpid behavior.fig . 5 a d 24-h echograms from the bottom - mounted 38 khz echo sounder on 8 august 2008 ( a ) , 18 october 2007 ( b ) , 2 february ( c ) , and 12 march 2008 ( d ) . the echograms are zoomed in at ~300390 m. the outlined boxes are presented in higher resolution in ( e h ) . the color scale refers to average volume backscattering ( s v ) ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset . time is given as utc ( local standard time 1 h ) a d 24-h echograms from the bottom - mounted 38 khz echo sounder on 8 august 2008 ( a ) , 18 october 2007 ( b ) , 2 february ( c ) , and 12 march 2008 ( d ) . the echograms are zoomed in at ~300390 m. the outlined boxes are presented in higher resolution in ( e h ) . the color scale refers to average volume backscattering ( s v ) ; dark red illustrates the strongest , and white the weakest backscatter . black and white bars above echograms depict night and day separated by times for sunrise and sunset . the average ts ( db ) of glacier lanternfish as measured between ~340 and 380 m were always in the range of approximately 57.5 to 59.5 db . within this limited range , the ts distribution of glacier lanternfish showed two peaks ( in both summers ) and decreased from the autumn to spring ( fig . 6average ts ( db ) of glacier lanternfish ( total n = 209,768 ) calculated from ~340 to 380 m for each month throughout the study period ( july 2007october 2008 ) average ts ( db ) of glacier lanternfish ( total n = 209,768 ) calculated from ~340 to 380 m for each month throughout the study period ( july 2007october 2008 ) the ts values were used in assessing the numerical densities of glacier lanternfish . the concentration of fish was usually in the range of 0.0050.015 individuals m ( fig . however , the maximum density , just below 0.02 individuals m , was recorded in july 2008 in the deepest interval ( 300390 m ) , and in october 2008 in the shallower interval ( 250300 m ) ( fig . 7 a , b estimated monthly average concentration ( individuals per m ) of glacier lanternfish during day and night from ( a ) 250300 m and ( b ) 300390 m during july 2007october 2008 . the white and black squares indicate the median daytime and nighttime concentration estimates , respectively . the black lines illustrate the 1st and 3rd quartile , while the x indicates the average estimated concentration a , b estimated monthly average concentration ( individuals per m ) of glacier lanternfish during day and night from ( a ) 250300 m and ( b ) 300390 m during july 2007october 2008 . the white and black squares indicate the median daytime and nighttime concentration estimates , respectively . the black lines illustrate the 1st and 3rd quartile , while the x indicates the average estimated concentration in spring and summer , when ndvm is the dominating mode of migration , the daytime concentration of the interval ~250300 m was higher than during night ( fig . below 300 m , where the nodvm mode were dominating , relatively little concentration fluctuations between day and night occurred , except for june august 2008 ( fig . 7b ) when the ndvm layers entered those depths at daytime ( fig . 2 ) . in the months where idvm were the dominating mode of migration ( i.e. september february ) , the concentration of glacier lanternfish decreased during daytime in the interval ~250300 m ( fig . 7a ) , as the inverse vertical migrators that were distributed from ~220 to 300 m during nighttime ascended to mid - waters ( in the lower part of the strong sl between ~150220 m ) during the day ( figs . 2 , 3 ) the 16-month acoustic data series revealed three different diel behavioral modes of glacier lanternfish . in ndvm , individuals ascend toward the surface at night and descend back to deeper waters in the morning . in idvm , individuals ascend toward mid - waters at daytime , before descending at night . in nodvm a clear seasonal pattern , with ndvm prevailing during spring and summer , and idvm prevailing during autumn and winter , was also apparent . all through the year , nodvm occurred in the water column deeper than ~300 m. this diversity in diel behavior of glacier lanternfish has previously been observed during autumn in masfjorden by kaartvedt et al . note , however , that while the acoustic results unveil the main patterns in diel behavior for the part of the population below ~200 m , they do not provide the entire picture for the whole population . only idvm was recorded acoustically in october and november , yet trawl catches documented some ndvm ( kaartvedt et al . also previous studies have documented ndvm of glacier lanternfish in fall and winter ( kaartvedt et al . 1988 ; giske et al . 1990 ; bagien et al . 2001 ) . some shallower - living glacier lanternfish intermingle with , and thus become inseparable in the acoustic signatures of the sl dominated by adult pearlside ( torgersen and kaartvedt 2001 ) . shallower - living glacier lanternfish are mostly smaller ( dypvik et al . 2012 ) and might be too small to be properly detected at 38 khz . the largely consistency in ts values in deep water underlines that we likely have addressed the adult population throughout this study period . the slight apparent seasonal variation in ts does not seem to correlate with behavioral changes . during spring and summer , , the concentration of zooplankton peaks in the surface layers , and significantly lowers in the deeper waters ( atkinson and peck 1988 ; rasmussen and giske 1994 ; richter 1994 ) . ndvm among lanternfishes is usually related to avoidance of visual predators in bright surface waters at daytime and subsequent feeding on zooplankton in the surface layers at night ( holton 1969 ; dalpadado and gjster 1987 ; kinzer et al . 1993 ) . the pay - off from ndvm will decrease as surface plankton becomes less abundant throughout fall and winter , while at the same time , seasonally migrating copepods return to depth ( speirs et al . 2006 ; broms and melle 2007 ) . . however , small individuals with less energy reserves , larger motivation , or less capability for feeding in deep water may continue to migrate toward the surface at night ( giske and aksnes 1992 ) , while larger fish may opt for other strategies such as staying in deeper waters feeding on overwintering calanoid copepods . in accordance with this , mainly 2-year or older glacier lanternfish are distributed deeper than ~250 m , while smaller individuals are distributed in shallower waters , during autumn ( dypvik et al . similar size - dependent depth distribution has also been observed in other studies of lanternfishes ( willis and pearcy 1980 ; gartner et al . idvm became apparent in august and was recorded until march . in winter , the bulk of zooplankton biomass is distributed in mid - waters ( giske et al . ( 2012 ) showed that inversely migrating glacier lanternfish were foraging on overwintering calanus in mid - waters during the day . other studies have shown that calanus is an important part of the glacier lanternfish diet during fall and winter ( gjster 1973 ; balio and aksnes 1993 ) . the seasonal occurrence of idvm seems to harmonize well with the overwintering period for calanus finmarchicus ( bagien et al . 2001 ; speirs et al . 2006 ) , in both the timing of initiation of this behavior , and its termination in spring ( heath 1999 ; astthorsson and gislason 2003 ; speirs et al . 2006 ; broms and melle 2007 ) . by staying in deeper waters performing idvm , glacier lanternfish may optimize their energy budget and reduce the predation risk ( dypvik et al . in addition , light decreases rapidly with depth in seawater ( srnes and aksnes 2006 ; aksnes et al . 2009 ) . a lanternfish swimming toward shallower waters , while still staying below ~200 m , will thus experience an increase of light in the surroundings , which would favor visual feeding by the low - light adapted lanternfish ( warrant and locket 2004 ) . thus , it seems likely that individuals with idvm ascend toward the overwintering layer of calanus at daytime to feed at favored light levels , sinking back to deeper layers at night . however , the proportion of the population with idvm decreases through the winter , which may reflect the reduced concentration of calanus from autumn through winter ( bagien et al . individuals of glacier lanternfish with nodvm occurred all year at depths greater than ~300 m. dypvik et al . ( 2012 ) found that the individuals with nodvm during autumn were the largest individuals of the glacier lanternfish population . individuals of greatest size tend to have larger eyes and might be better adapted than smaller individuals to detect prey in low light ( warrant and locket 2004 ) . they are also better adapted for handling large prey such as krill and shrimps , which occur deep in the water column ( kaartvedt et al . 2012 ) , and which represent a much higher dividend than copepods due to their much larger size ( falk - petersen 1981 ; tande 1982 ) . we therefore suggest that individuals with nodvm are able to feed in the restricted light levels of the deep waters . the behavior of individuals in deep water ( > 300 m ) suggested that the nodvm group indeed largely consisted of non - migrating individuals , and that consistent backscatter in deep water is not a result of asynchronous migrations , but that the population rather splits into migrating and non - migrating individuals , as initially suggested by pearcy et al . ( 1979 ) . nevertheless , single individuals were observed swimming upwards or downwards in a step - wise fashion , as also observed by kaartvedt et al . intermittent vertical relocations , ascent / descent in a step - wise fashion , would help the fish scan different parts of the water column for prey . fish may also use their lateral lines for tactile prey detection in darkness ( janssen et al . 1999 ) , and such behavior would be in accordance with both visual ( obrien et al . 5b ) suggests a prominence of ascent at day , so we can not reject the possibility that some of these deep - living individuals took part in idvm at that time . yet , most step - wise movements seemed to represent infrequent re - locations within the layer . a wide variety of lanternfishes are capable of feeding at depths greater than several hundreds of meters ( pearcy et al . 1979 ; roe and badcock 1984 ; sameoto 1988 ) , and nodvm has previously been observed among several species of lanternfish ( pearcy et al . however , different species of calanus occur in different geographic regions and , as well as other genera , perform seasonal vertical migration , overwintering in deeper water ( atkinson and peck 1988 ; richter 1995 ; peterson 1998 ; broms and melle 2007 ) . we therefore hypothesize that idvm and nodvm are widespread behaviors in areas where concentrations of potential prey are distributed in deeper waters , and that similar plasticity in dvm behavior as observed for glacier lanternfish in masfjorden might occur in other populations of lanternfishes in areas with seasonally migrating zooplankton , such as overwintering calanoid copepods . long - term acoustic measurements might be used to reveal such dvm plasticity in other areas , information that could be used to gain knowledge about interactions between lanternfishes , their prey and predators , as well as lanternfishes contribution to the vertical flux of organic matter . the vertical distribution and diel vertical migration of lanternfishes can affect such interactions and processes , making it imperative to further reveal these patterns . acute liver failure ( alf ) can occur as a result of various etiologies including hepatic injury by drugs and poison , viral hepatitis , ischemia , or other causes . the mechanisms by which liver cells are destroyed as well as the processes mediating liver regeneration , remain largely unknown . it has become evident that liver cell death can occur via distinct biochemical pathways and morphological alterations , including apoptosis , autophagic cell death , and necrosis . apoptosis is defined by chromatin condensation , nuclear fragmentation , cell shrinkage , blebbing of the plasma membrane , and formation of apoptotic bodies that contain nuclear or cytoplasmic material ( kerr et al . , 1972 ; autophagic cell death , on the other hand , is characterized by a massive accumulation of double - membrane containing vacuoles , called autophagosomes , that subsequently fuse with lysosomes . necrotic cell death is often negatively defined as a form of cell death that lacks signs of apoptosis or autophagy . typically , necrotic cells show cytoplasmic swelling , dilation of organelles , and mechanical rupture of the plasma membrane . although necrosis has been deemed to be a mainly passive process , the initiation , and modulation of necrotic cell death are currently under intense investigation at the molecular level . the relative contribution of apoptosis or necrosis to organ dysfunction in alf remains controversial ( schulze - osthoff and bantel , 2011 ) . necrosis is typically the consequence of acute metabolic perturbation with atp depletion , whereas apoptosis represents an atp - dependent cell death program . furthermore , in several cases , the nature and duration of cellular injury determine if cells die by apoptosis , necrosis , or other mechanisms . at low doses , a variety of injurious stimuli often induce apoptosis , but the same stimuli can result in necrosis at higher doses . therefore , in many situations cell death might be not executed as a clear - cut form of cell death , but as a continuum with intermediate features of both apoptosis and necrosis . a distinction of different cell death forms is therefore not only relevant for semantical reasons , but has important clinical implications when considering the therapeutic targeting of cell death processes . thus , an understanding of the cell death processes is most important for development of effective interventions to prevent hepatocellular death in acute liver damage ( fischer and schulze - osthoff , 2005 ) . apoptosis represents a programmed form of cell death that is required for the maintenance of tissue homeostasis by counterbalancing cell proliferation and eliminating damaged , infected , or transformed cells . this process is particularly important in the liver as an organ that is naturally exposed to toxins , drugs , and viruses . apoptosis results from a collapse of cellular infrastructure through internal proteolytic digestion , which leads to cytoskeletal disintegration , metabolic derangement , and genomic fragmentation . members of the caspase family of proteases form the core engine of apoptosis and are involved in initiation , execution , and regulatory phases of the pathway . caspases are cysteine proteases that cleave substrates after aspartate residues within specific peptide recognition sequences . to preclude unwarranted cell death , caspases are expressed as inactive zymogens consisting of a prodomain followed by two subunits with the catalytic domain . caspases operate in hierarchical cascades that serve to amplify the apoptotic signal ( los et al . , 1999 ) . based on their structure and order in cell death pathways effector caspases such as caspase-3 , -6 , and -7 cleave diverse cellular substrates including structural proteins such as cytokeratin-18 and many others ( fischer et al . , in contrast , initiator caspases , such as caspase-8 , -9 , and -10 , exert regulatory roles by activating downstream effector caspases . caspases are activated by two major signaling routes , namely the extrinsic death receptor and the intrinsic mitochondrial pathway , that both depend on the formation of large multi - protein complexes ( schulze - osthoff et al . , 1998 ; initiator caspase-8 is the key mediator of the extrinsic pathway . in a simplified model , binding of death ligands such as trail or cd95l or this event then results in the recruitment of the adapter protein fadd and the initiator caspase-8 into a death - inducing signaling complex ( disc ) , wherein caspase-8 becomes activated by dimerization and autoproteolytic cleavage ( figure 1 ) . subsequently , caspase-8 cleaves and activates the effector caspase-3 , culminating in the demise of so - called type i cells . in most cells including hepatocytes , however , only low amounts of initiator caspases are activated at the disc , which is insufficient for cell death . in those type ii cells , the extrinsic receptor pathway must be amplified by the intrinsic mitochondrial apoptotic pathway through the caspase-8-mediated cleavage of bid , a pro - apoptotic bcl-2 family protein , which subsequently initiates together with the bcl-2 family members bak and bax the release of mitochondrial pro - apoptotic mediators ( schwerk and schulze - osthoff , 2005 ) . interestingly , cd95-induced hepatocyte apoptosis is delayed but not inhibited in bak / bax - deficient mice indicating that hepatocytes can act as type i cells in the absence of pro - apoptotic bcl-2 proteins ( hikita et al . , activation of nf-b is mediated by traf-2 , rip-1 , and other signaling molecules that lead to activation of ib kinase and subsequent activation of nf-b target genes . fadd and caspase-8 are the essential adapter proteins involved in apoptosis , which in hepatocytes requires a mitochondrial amplification loop through caspase-8-mediated cleavage of bid . the subsequent translocation of bax and bak results in mitochondrial outer membrane permeabilization , cytochrome c release , and effector caspase-3 activation . under conditions of impaired apoptosis , tnf receptor-1 can induce necroptosis , which involves rip-1 and rip-3 kinases . among other effects , rip-3 can increase the production of reactive oxygen species ( ros ) due to increased oxidative phosphorylation , resulting in intracellular calcium overload , mitochondrial membrane permeability transition ( mpt ) , depletion of atp , and necrosis . apap - induced necrosis is essentially mediated by a toxic metabolite , which depletes glutathione and forms apap protein adducts , triggering oxidative stress , compromised respiratory function , and atp depletion . although apap treatment can instigate the mitochondrial pathway of apoptosis , high doses of apap will ultimately mediate liver cell death by necrosis . this has been impressively demonstrated in mice that died rapidly of liver failure with massive hepatocyte apoptosis when agonistic anti - cd95 antibody was injected ( ogasawara et al . , 1993 ) . similarly , treatment of mice with tnf- in combination with a transcription - blocking agent , such as d - galactosamine ( d - galn ) or actinomycin d , induces lethal hepatitis ( leist et al . , 1994 , 1995 ; another well established mouse model of alf consists of a combined treatment with d - galn and lipopolysaccharide ( lps ) , which induces tnf- expression and an inflammatory response that is predominantly directed toward the liver ( galanos et al . , 1979 ) . it has been suggested that the toxicity in the murine tnf- and anti - cd95 models resembles viral forms of acute hepatic failure in patients ( keppler et al . , 1968 ; el - mofty et al . , 1975 ) . cd95/cd95l expression has been shown to be upregulated in viral hepatitis and to correlate with disease activity and hepatocyte apoptosis ( hiramatsu et al . , 1994 ; mita et al . , 1994 ; pianko et al . , 2001 ; lee et al . , we have recently demonstrated that diseased , e.g. , hcv - infected , livers show an upregulation of trail receptors and increased susceptibility toward trail - induced apoptosis ( volkmann et al . , 2007 ) . these data implicate that viral forms of alf are associated with death receptor - induced cell death . recent data show that the cd95 system is involved in human alf caused not only by viral hepatitis but also by wilson s disease ( strand et al . , 1998 ; increased levels of death ligands or receptors such as cd95l , tnf- , or tnf receptors ( tnf - r ) were found in blood of patients with alf ( ryo et al . particularly , high serum levels of soluble death receptor cd95 have also been found in drug - induced alf ( tagami et al . , 2003 ; rutherford et al . , 2007 ) . silencing of cd95 or caspase-8 protected mice from alf or fulminant hepatitis induced by agonistic cd95 antibody or concanavalin a , respectively ( song et al . however , cd95 and caspase-8 also promotes liver regeneration by inducing differentiation of stellate cells and possibly of other non - parenchymal liver cells ( desbarats and newell , 2000 ; canbay et al . , 2003 ; ben moshe et al . , 2007 ) . additionally , tnf- plays a pivotal role in liver regeneration by activation of transcription factors such as nf-b , which induces the transcription of a huge number of cytokines and growth - promoting target genes ( wullaert et al . , 2007 ) . the activation of nf-b by tnf- is mediated by distinct adapter proteins that are recruited to tnf - r-1 upon ligand binding ( figure 1 ) . whether increased levels of circulating death receptors or ligands in human alf mirror apoptotic cell death or liver regeneration and whether death receptor - induced cell death depends on special alf etiologies remains unknown . in contrast to viral infection , drug - induced liver injury is mainly associated with signaling pathways triggered by mitochondrial damage ( chan et al . , 2005 ) . in the intrinsic pathway , apoptosis is mediated by translocation of pro - apoptotic bcl-2 molecules , such as bax and bak , from the cytosol to mitochondria to form pores in the outer mitochondrial membrane ( los et al . , 1999 ) . this process is followed by the mitochondrial release of cytochrome c and other pro - apoptotic factors . cytochrome c normally functions in electron transport processes of the respiratory chain to generate atp . in the cytosol of apoptotic cells , however , it serves as a cofactor for the adapter protein apaf-1 . upon binding of cytochrome c and datp , apaf-1 oligomerizes , and recruits the initiator caspase-9 to trigger the formation of the apoptosome thus , similar to the disc , the apoptosome is a high - molecular weight complex that serves as a caspase activation platform . once assembled in the apoptosome , caspase-9 becomes activated and subsequently triggers the caspase cascade ( schulze - osthoff et al . as mentioned above , there is also considerable crosstalk between the extrinsic and intrinsic pathways . for example , during death receptor - mediated apoptosis caspase-8 can proteolytically activate the bcl-2 protein bid , which facilitates cytochrome c release and amplifies the apoptotic signal following death receptor activation . in contrast to apoptosis , necrosis is mediated by opening of the mitochondrial membrane permeability transition ( mpt ) pore , which triggers the collapse of the membrane potential and cessation of atp formation . the resulting mitochondrial swelling leads to the rupture of the outer mitochondrial membrane with the release of intermembrane proteins and subsequent nuclear dna fragmentation . other prominent features include massive energy depletion , formation of reactive oxygen species ( ros ) , and activation of non - apoptotic proteases . the elevated calcium levels in the cytosol trigger mitochondrial calcium overload , leading to depolarization of the inner mitochondrial membrane and a shut - down of atp production . while depletion of atp impedes the function of membrane channels , increased calcium activates calcium - dependent proteases , such as calpains . calcium fluxes , atp depletion , and oxidative stress involve complex and interactive feedback loops , which self - amplify and potentiate each other leading to exaggerated cell death . the relative amount of atp might be an important factor that determines whether hepatocytes die by apoptosis or necrosis ( ferrari et al . when the necrotic cell ruptures , an inflammatory response follows due to the release of intracellular contents . in contrast , inflammation is not typical of apoptosis , because phagocytic cells rapidly engulf apoptotic cells and thereby prevent the release of noxious intracellular compounds . acetaminophen ( paracetamol , n - acetyl - p - aminophenol ; apap ) overdose represents one of the most common causes of alf in developed countries ( larson et al . , 2005 ) . apap - induced hepatotoxicity is due to the formation of the toxic metabolite n - acetyl - p - benzoquinone imine by the cytochrome p450 system , which causes glutathione depletion , oxidative stress , alterations of calcium homeostasis , resulting in mpt , loss of mitochondrial membrane potential , and atp depletion ( hinson et al . , 2010 ) . although necrosis has been thought to be the predominant mode of cell death in apap - induced liver injury , conflicting in vitro and animal data have emerged suggesting a potential role of apoptosis in acetaminophen - induced hepatotoxicity ( el - hassan et al . mice treated with a toxic dose of acetaminophen showed 40% apoptotic and 60% necrotic hepatocytes ( ray et al . , 1996 ) . it was demonstrated that mice with defective cd95 receptor ( lpr mice ) or cd95 ligand ( gld mice ) were partially protected from apap - induced liver injury ( liu et al . , 2004 ) . moreover , increased circulating levels of cd95 have been found in humans with apap intoxication ( tagami et al . , 2003 ) . it has been recently suggested that apap hepatotoxicity is caused by the mitochondrial apoptosis pathway and facilitated by chemokine ( c - x - c motif ) receptor 2 ( cxcr2 ) signaling . in this study caspase inhibition prevented dna fragmentation , although the authors did not investigate whether caspase inhibition was also associated with cell survival ( hu and colletti , 2010 ; schulze - osthoff and bantel , 2011 ) . in contrast , in another study no caspase activation was observed and , accordingly , caspase inhibition did not protect from liver injury in apap - treated mice ( jaeschke et al . , 2006 ) . further data from animal models revealed also no evidence that apoptotic cell death contributes to apap - induced liver injury . for instance , following the application of an apap overdose in mice , less than 1% of the parenchymal cells revealed an apoptotic morphology ( gujral et al . , 2002 ) . another study in mice showed that knockdown of the cd95 receptor protected against 300 mg / kg apap overdose but not against 700 mg / kg overdose ( zhang et al . , 2000 ) , indicating that the mode of cell death might at least partially depend on the apap dose . indeed , several studies demonstrated that apap induces mitochondrial dysfunction with atp depletion which even interrupts initial cd95-induced mitochondrial signaling pathways ( lawson et al . on the other hand , subliminal cd95 activation can also increase apap - induced liver injury ( tinel et al . , 2004 ) , indicating that death receptor signaling might influence the extent of apap - induced necrotic liver injury . conflicting data exist also about the role of tnf- in apap - induced liver injury . increased tnf- expression in liver and circulating tnf- levels have been observed after apap poisoning ( blazka et al . . however , the role of tnf- in apap - induced necrosis remains controversial , as tnf- inhibitors exerted either protection or no effect ( blazka et al . , 1995 ; simpson et al . in addition , tnf- knockout mice showed similar sensitivity to acetaminophen compared to wildtype mice ( boess et al . , 1998 ) . it has been recently shown that inhibition of c - jun n - terminal kinase ( jnk ) , a member of the mitogen - activated protein kinase family , reduced paracetamol - induced toxicity in mice by inhibiting hepatic tnf- production ( henderson et al . , 2007 ) . furthermore , apap - induced jnk activation has been linked to activation of the pro - apoptotic bcl-2 protein bim . in line , apap - induced necrotic liver injury was shown to be reduced in bim knockout mice ( badmann et al . , however , neither jnk1 nor jnk2 knockdown did protect mice from apap - induced liver toxicity , raising concerns about a major role of jnk in apap - induced liver injury ( gunawan et al . , 2006 ; henderson et al . , 2007 ; bourdi et al . , 2008 ) . altogether , although apoptotic alterations can occur , profound energy depletion , and mitochondrial failure presumably divert cell death to necrosis as the principal mode of apap - induced liver toxicity . autophagy represents another process that might influence the outcome of apap - induced liver toxicity . autophagy is a catabolic mechanism by which long - lived proteins and organelles are recycled in order to maintain energy and protein synthesis . it is characterized by the appearance of numerous cytosolic vacuole - like structures , called autophagosomes , which encapsulate cytosolic materials and fuse with lysosomes . although the role of the autophagy in protection during nutrient starvation is accepted , its function in programmed cell death remains controversial . under normal physiological conditions autophagy occurs at low basal levels , contributing to the turnover of cytoplasmic components and promoting cell survival during stress conditions . interestingly , it has been recently demonstrated that apap induces autophagy in mouse liver and primary human hepatocytes and that activation of autophagy protects against apap - induced hepatotoxicity ( ni et al . , it was suggested that the induction of oxidative stress might play an important role in apap - induced autophagy . moreover , it was shown that pharmacological inhibition of autophagy increased apap - induced liver injury . clearly , more work is needed to elucidate the role of autophagy in apap - induced hepatotoxicity and the complex crosstalk with other cell death pathways . in addition to measuring death ligands , several other cell death biomarkers have been proposed for monitoring the clinical outcome of alf and other liver diseases ( volkmann et al . , 2006 ; rutherford et al . caspases cleave the intermediate filament protein cytokeratin ( ck)-18 into specific fragments that are released into circulating blood and can be detected by the m30 elisa ( bantel et al . , 2001b , 2004 ; seidel et al . , 2005 ) . moreover , when this assay is combined with a second elisa that detects the total release of caspase - cleaved and uncleaved ck-18 ( m65 elisa ) , even different forms of cell death , such as necrosis and apoptosis , can be discriminated . using these serological assays , it has been recently demonstrated that the predictive sensitivity of total ck-18 for lethal outcome was comparable to the model for end - stage liver disease ( meld ) score at time of admission of alf patients ( bechmann et al . , 2010 ) . moreover , modification of the meld score by substitution of bilirubin for total ck-18 significantly increased the prediction of alf outcome . interestingly , we have demonstrated that alf patients display considerable caspase activity and high levels of caspase - cleaved ck-18 in the serum , which was unexpectedly higher in spontaneous survivors than in patients that required transplantation or died ( volkmann et al . , 2008 ) . nevertheless , despite a weaker activation of caspases , liver biopsies of patients without spontaneous recovery revealed extensive tunel reactivity , which detects both apoptotic and necrotic cell death . moreover , sera from those patients contained increased levels of total ck-18 , but reduced levels of its caspase - generated fragments as compared to patients with spontaneous recovery . these findings therefore indicate that necrosis but not apoptosis is the predominant cell death in those critically ill alf patients , whereas in alf patients with spontaneous recovery apoptotic cell death predominates . in contrast , in another study , detection of caspase - cleaved ck-18 could not adequately predict alf outcome ( rutherford et al . however , unlike in our study in which apap - induced alf only played a minor role , in this study a relevant number of patients with apap - induced alf was included . as mentioned above , in those patients necrotic cell death might predominate irrespective of the outcome . in line with this observation , patients with apap - induced alf showed higher levels of total ck-18 compared to caspase - cleaved ck-18 levels ( bechmann et al . 2008 ; craig et al . , 2011 ; bantel and schulze - osthoff , 2012 ) . caspase activation might also play a role in liver regeneration and this might explain our observation of higher caspase activity in spontaneous survivors . caspases can cleave different cytokine precursors to generate active cytokines and thus create an environment that could be essential for liver regeneration . experiments in mice showed that caspase activation is associated with chemokine production and inflammation in the liver , whereas caspase-3 inhibition strongly reduced activity of pro - inflammatory transcription factors and chemokines ( faouzi et al . , 2001 ) . a role of caspases in liver regeneration is best exemplified by a recent report showing that a hepatocyte - specific knockout of caspase-8 attenuates hepatocyte proliferation after partial hepatectomy ( ben moshe et al . , 2007 ) . in addition to cell death biomarkers , circulating levels of nucleosomes and high - mobility group box 1 ( hmbg1 ) protein , both of which are released during hepatocyte death , have been shown to be elevated in patients with alf 2011 ) . however , those biomarkers were not able to predict alf outcome , and no significant differences in hmbg1 or nucleosome levels between paracetamol- and non - paracetamol - induced liver injury were found ( craig et al . , 2011 ) . one explanation for the lacking correlation of those biomarkers with alf outcome might be that the release of hmbg1 and nucleosomes are not strictly related to necrotic cell death , but also occur during apoptosis ( bell et al . certainly , further larger cohort studies are required to evaluate the predictive value of cell death markers in patients with paracetamol- or non - paracetamol - induced alf . an admixture of necrosis and apoptosis occurs in alf and might therefore open up novel strategies for therapeutic intervention ( fischer and schulze - osthoff , 2005 ) . more extreme injury leads to necrotic killing , whereas milder injury may result in apoptosis . whether inhibition of apoptosis using available pharmacological caspase inhibitors can indeed prevent liver cell death or will just simply shift the mode of cell death to necrosis , remains to be shown ( los et al . , 2002 ) . as caspases not only play a role in apoptosis but also in processes of liver regeneration , the possibility of adverse effects should not be ignored when considering the therapeutic use of caspase inhibitors in alf treatment . death receptor - blocking agents , such as cd95-fc or trail - r - fc proteins , might be promising candidates in virus - induced alf ( bantel and schulze - osthoff , 2003 ) . micrornas ( mirnas ) are non - coding rnas which have been implicated in the posttranscriptional regulation of various cellular pathways . it was recently demonstrated that overexpression of mirna-221 delays cd95-induced fulminant liver failure in mice ( sharma et al . , 2011 ) . thus , mirnas involved in cell death signaling pathways might serve as potential therapeutic targets in alf . due to the pleiotropic effects of tnf- , inhibitors of this cytokine might not only influence liver cell death but also immune response and liver regeneration . treatment of apap intoxicated mice with anti - tnf- partially prevented hepatotoxicity ( blazka et al . , other studies showed no alterations of apap toxicity in mice treated either with anti - tnf- antibody or soluble tnf receptor ( simpson et al . whether the observed discrepancy is due to the variant experimental conditions , such as different mouse strains , remains to be elucidated ( jaeschke et al . , there is increasing evidence suggesting that tnf- triggers cell death not only by apoptosis , but also by a necrosis - like process , which has been recently called necroptosis ( schulze - osthoff et al . whereas tnf--induced apoptosis involves caspase-8 , tnf--induced necroptosis is essentially mediated by the kinases rip-1 and rip-3 that are recruited to tnf - r1 ( figure 1 ) . interestingly , specific inhibitors of rip-1 , called necrostatins , have been recently designed as novel cytoprotective agents that might be a promising tool to positively influence alf outcome , at least in conditions in which death receptor - mediated necrosis predominates . therapeutic targets to improve apap - induced alf outcome might be jnk or other members of the mitogen - activated protein kinase family , such as p38 . jnk inhibition by sp600125 , a small - molecule reversible atp - competitive inhibitor , or by d - jnkii , a peptide inhibitor that inhibits the interaction of jnk with substrates , markedly reduced mortality in murine paracetamol - induced hepatotoxicity , with a significant reduction of hepatic apoptosis and necrosis ( bennett et al . , 2001 ; borsello et al . , 2003 ; henderson et al . , 2007 ) . since glutathione depletion and subsequent oxidative stress formation are key pathogenic mechanisms of apap - induced hepatocyte death , application of n - acetylcysteine , a pro - drug for glutathione synthesis , which has been shown to reduce apap - induced liver necrosis , is the current standard therapy in apap - induced alf ( corcoran et al . , 1985 ; saito et al . , 2010 ) in addition , other drugs with anti - oxidant effects might improve alf outcome . in this respect , application of cyclooxygenase inhibitors exerted protective effects in an experimental alf mouse model with decreased oxidative stress formation and marked reduction of hepatic necrosis ( chang et al . a novel therapeutic target in alf that connects the immune system with cell death might be cyclophilin a. this intracellular protein exerts pro - inflammatory and hepatotoxic activity when released from necrotic liver cells in apap - induced liver injury . conversely , inhibition of cyclophilin reduced inflammatory response to necrotic liver ( dear et al . , 2011 ) . thus , targeting of cyclophilin might represent an opportunity for a novel therapeutic approach in acetaminophen poisoning . as mentioned above , pharmacological inhibition of autophagy exacerbated apap - induced liver toxicity . vice versa , induction of autophagy by rapamycin was shown to inhibit apap - induced hepatotoxicity ( ni et al . , 2012 ) . thus , autophagy induction might be a further strategy to improve alf outcome in apap intoxicated patients . taken together , there are many promising therapeutic approaches for inhibition of apap- and non - apap - induced alf . however , it should be kept in mind that most of the described targets exert a pleiotropic role and might interfere not only with cell death but also with survival pathways . 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 seasonal variations in glacier lanternfish ( benthosema glaciale ) vertical distribution and diel vertical migration ( dvm ) were studied by use of a bottom - mounted upward - facing 38 khz echo sounder deployed at 392 m depth and cabled to shore in masfjorden ( ~6052n , ~524e ) , norway . acoustic data from july 2007october 2008 were analyzed , and scattering layers below ~220 m during daytime were attributed to glacier lanternfish based on net sampling in this , and previous studies , as well as from analysis of the acoustic data . at these depths , three different diel behavioral strategies were apparent : normal diel vertical migration ( ndvm ) , inverse dvm ( idvm ) , and no dvm ( nodvm ) . nodvm was present all year , while idvm was present in autumn and winter , and ndvm was present during spring and summer . the seasonal differences in dvm behavior seem to correlate with previously established seasonal distribution of prey . we hypothesize that in regions with seasonally migrating zooplankton , such as where calanoid copepods overwinter at depth , similar plasticity in dvm behavior might occur in other populations of lanternfishes . acute liver failure ( alf ) can be the consequence of various etiologies , that might vary between different geographic regions . most frequent are intoxications with acetaminophen , viral hepatitis , or liver damage of unknown origin . alf occurs when the extent of hepatocyte death exceeds the regenerative capacity of the liver . the mode of liver cell death that is predominantly induced in alf , i.e. , apoptosis or necrosis , is still controversial and presumably determined by the etiology , duration , and magnitude of liver injury . severe liver damage involves oxidative stress and depletion of atp resulting in necrosis . in contrast , maintenance of atp stores is required for the execution of apoptosis . recent data suggest that necrosis resulting from severe liver damage is associated with poor outcome of alf patients . discrimination between apoptosis and necrosis might be therefore useful for the identification of alf patients requiring liver transplantation . identification of the molecular cell death mechanisms remains an important issue not only for early prediction of alf outcome , but also for therapeutic interventions . in view of the pleiotropic functions of critical mediators of cell death and tissue regeneration , a particular challenge will be to reduce hepatocellular death without inhibiting the regenerative capacity of the liver . here , we review the molecular mechanisms of hepatocyte injury and the pathways leading to apoptosis and necrosis , which might represent potential diagnostic and therapeutic targets in alf .

age - related macular degeneration ( amd ) is subdivided into the dry form and the wet form . according to bressler et al . the minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular age - related macular degeneration ( marina ) evaluated the benefit of ranibizumab in a dose of 0.5 mg per month . at 12 months , 95% of patients had lost < 15 early treatment diabetic retinopathy study ( etdrs ) letters from baseline and 34% of patients had a best corrected visual acuity ( bcva ) gain of 15 etdrs letters . the study ranibizumab in patients with subfoveal choroidal neovascularization ( cnv ) secondary to age - related macular degeneration ( sustain study ) was designed to further evaluate the safety , tolerability , and efficacy of optical coherence tomography ( oct)/bcva - guided , individualized , and flexible pro re nata ( prn , as needed ) dosing regimen for ranibizumab . at 12 months , 92.5% of patients had lost < 15 etdrs letters from baseline and 19.3% of patients had a bcva gain of 15 etdrs letters . whether the fixed or prn regimen was used in the treatment of wet amd , the therapeutic response of individual patients was not always the same . current observations suggest that the factors which affect the response to the initial treatment with ranibizumab are individual . the aim of this study was to assess the importance of various factors ( age , gender , baseline bcva , baseline macula thickness , and type and size of cnv ) for early morphological therapeutic response to ranibizumab in clinical practice . the treatment of patients with wet amd is centralized into 9 tertiary referral centres in the czech republic ( see appendix ) . anonymised data on treatment efficacy and safety have been consecutively entered into the czech national database amadeus ( age - related macular degeneration in patients in the czech republic ) since september 2008 . the main aim of this registry is to collect basic epidemiologic data on patients diagnosed with wet amd in the czech republic , document standard diagnostic and therapeutic patterns , and assess treatment efficacy in standard clinical practice . the data collection is independent of all treatment decisions ; it does not affect a patient 's access to treatment and fully complies with all ethical as well as legal requirements for noninterventional data collection in the czech republic . all patients have given written informed consent to the treatment , as well as data collection . the reported investigations were in accordance with the principles of the current version of the declaration of helsinki . the data are recorded from the moment of diagnosis and start of treatment at regular 3-month intervals for half a year . in the following period , the record is completed every 6 months . each record presumes biomicroscopic examination of the retina , determination of bcva using the etdrs chart , and an oct examination ( oct 3 stratus ) . the first visit involves fluorescein angiography ( fa ) ; indocyanine green angiography ( icga ) is used only if it is necessary for determining a diagnosis . the compulsory data always include bcva expressed by the number of etdrs letters , the central thickness of macula in 1 mm of macula in m , and volume in 6 mm of macula in mm . the first visit also involves recording age and gender and measuring type and size of cnv using fa or icga . patients with diagnosed wet form of amd who meet the czech society of ophthalmology criteria for initiation of treatment with the ranibizumab are entered into the registry . ranibizumab therapy in the czech republic is indicated in patients with amd who are older than 50 years , with predominantly classic , minimally classic , or occult cnv in subfoveal localization , a bcva score between 70 and 35 letters ( 20/4020/200 snellen equivalent ) , total macular lesion area 8 disc area ( da ) , and submacular haemorrhage 25% of the total macular lesion area . minimally classic and occult cnv must show signs of activity in the form of the presence of hard exudates , subretinal haemorrhages , or decrease in bcva within the last 3 months by 10 letters of the etdrs chart . in patients treated with ranibizumab in a dose of 0.5 mg , there are two separate phases : the loading phase , followed by a prn phase . in the loading phase , patients receive 3 consecutive monthly injections of ranibizumab ( months 02 ) , followed by a prn phase when further treatment is given between and including months 3 and 11 according to the retreatment criteria . retreatment with ranibizumab is performed if the patient 's bcva worsened against bcva recorded in the previous visit and if there is a demonstrable macular edema on oct examination . the prn method of application is also followed in the second year and all succeeding years of patient treatment . our study assessed the influence of age , gender , baseline bcva , baseline macula thickness , and type and size of cnv on early morphological therapeutic response to ranibizumab in clinical practice . we studied these factors in terms of anatomical changes in the macula after 3 consecutive monthly injections of ranibizumab in the loading phase ( months 02 ) . the monitored factors : age , gender , baseline best corrected visual acuity , baseline macula thickness , and type and size of choroidal neovascularization nor any others parameters ( visual acuity , oct ) had no influence on the treatment scheme in the loading phase . from 01/09/2008 to 24/6/2013 , 1153 newly diagnosed , treatment - nave patients treated with ranibizumab were entered into the registry . following the 3 initial injections of ranibizumab , the patients were divided into two groups based on the morphological findings in the macula : a group with active cnv and a group with inactive cnv . in the group with active cnv , oct screening revealed intraretinal macular edema , subretinal fluid accumulation , retinal pigment epithelium ( rpe ) detachment , fibrovascular rpe detachment , or the combination of all these findings . oct screening revealed restored foveal depression and a scar at the site of cnv with no signs of exudation was apparent . no intraretinal macular edema , subretinal fluid accumulation , rpe detachment , or fibrovascular rpe detachment was found . in both groups , we assessed the following parameters : gender and age of patients , type and size of cnv , baseline bcva on the etdrs chart , and baseline macular thickness . standard descriptive statistics were applied in the analysis : absolute and relative frequencies for categorical variables and median supplemented with 5th95th percentiles and mean supplemented by 95% confidence interval for continuous variables . the statistical significance of differences between groups was analyzed using pearson 's chi - square test for categorical variables and mann - whitney u test for continuous variables. = 0.05 was adopted as the level of statistical significance in all analyses . ( spss , inc . 2010 ) and performed by the institute of biostatistics and analyses at masaryk university , brno , operating independently of any amd treating centre in the czech republic . the sample included 1092 patients , 38.6% men , average age 73.3 years ( sd : 8.4 ) , and 61.4% women , average age 74.2 years ( sd : 8.6 ) . the analysis included 1153 treated eyes ; the right eye was treated 561 times , the left eye 592 times ( both eyes were treated 61 times ) . after the initial 3 injections ( day 0 , month 1 , and month 2 ) of ranibizumab , in month 3 we examined the sample of 1153 eyes . of these there were 841 eyes with active cnv ( the active group ) and 312 eyes with inactive cnv ( the inactive group ) . the sample in the inactive group included 41% of men and 59% of women ( p = 0.338 , pearson 's chi - square test ) . the active and the inactive group included 29.3% and 27.9% of patients at the age < 70 years , 43.4% and 42.9% of patients aged 7080 years , and 27.3% and 29.2% of patients at the age > 80 years ( p = 0.237 , mann - whitney u tests ) , respectively . the active and the inactive group included 31% and 20.2% of patients with predominantly classic cnv , 21% and 19.9% with minimally classic cnv , and 47.9% and 59.9% with occult cnv , respectively . the inactive group showed statistically significantly higher presence of occult membranes and statistically significant lower presence of predominantly classic cnvs compared with the active group ( p < 0.001 , pearson 's chi - square test ) . the active and the inactive group included 23.8% and 26.9% of patients with cnv < 2 disc areas ( da ) , 66.8% and 70.2% of patients with cnv 25 da , 9.4% and 2.9% of patients with cnv > 5 da , respectively . the inactive group showed statistically significantly lower presence of cnv > 5 da compared with the active group ( p < 0.001 , pearson 's chi - square test ) . the baseline bcva in the range of 1530 etdrs letters was shown in 10.2% of patients in the active group and 10.9% of patients in the inactive group . the bcva in the range of 3160 etdrs letters was shown in 62.5% of patients in the active group and 55.1% of patients in the inactive group . the baseline bcva of > 60 etdrs letters was shown in 27.2% of patients in the active group and 34% of patients in the inactive group . the median of the baseline bcva was 54 ( 595 percentiles : 2273 ) and 55 ( 595 percentiles : 2375 ) in the active and inactive group , respectively . we found no statistically significant difference in the value of the baseline bcva between the groups ( p = 0.066 , mann - whitney u test ) . 19.3% of patients in the active group and 15.6% of patients in the inactive group had a baseline macular thickness of < 250 m . the baseline macular thickness in the range of 250400 m was shown in 51.7% of patients and 58.5% of patients , respectively , and baseline macular thickness of > 400 m was shown in 29% and 25.9% of patients , respectively . the median of the baseline macular thickness was 330 m ( 595 percentiles : 190600 ) and 337 m ( 595 percentiles : 201535 ) in the active and inactive group , respectively . there was no statistically significant difference in the value of the baseline macular thickness between groups ( p = 0.663 , mann - whitney u test ) . in the literature , we found no publications describing prognostic factors for early morphological therapeutic response to treatment with ranibizumab in patients with wet amd . sarks , killingsworth , and gonzales noted that occult cnv may have a good functional treatment response . and killingsworth demonstrated histologically that the onset of cnv is characterized by intrachoroidal neovascularization followed by sub - rpe fibrovascular proliferation . occult cnv which is fibrovascular tissue in the sub - rpe space may in part represent an earlier stage of cnv because it is in the same tissue plane . up to 50% of occult cnv occult cnv as an earlier stage of the disease is assumed to be associated with less damage to photoreceptors in macula and successful treatment has a better prognosis . in our study , we evaluated the impact of gender , age , baseline bcva , baseline macular thickness , and type and size of cnv on early morphological therapeutic response following the 3 initial injections of ranibizumab . in the inactive group of 312 patients with complete regression of cnv activity after the initial 3 injections of ranibizumab , we found a statistically significantly higher proportion of occult membranes , statistically significant lower presence of predominantly classic cnv ( p < 0.001 ) , and statistically significantly lower incidence of cnv > 5 da ( p < 0.001 ) compared with the active group . we observed that smaller and occult cnv lesions have potentially better morphological therapeutic response with the disappearance of the cnv activity and resorption of the macular edema . we found no significant impact of gender , age , value of baseline bcva , or baseline macular thickness on early morphological therapeutic response after the initial 3 injections of ranibizumab . additional studies are needed to further clarify the relationship of morphological and functional results in ranibizumab treated patients with wet age - related macular degeneration . the results showed positive early morphological therapeutic response with restored foveal depression and no signs of exudation on oct in patients with higher incidence of occult cnv , lower incidence of predominantly classic cnv , and lower incidence of cnv > 5 da . there was no evidence of any effect of age , gender , baseline best corrected visual acuity , or baseline macula thickness on the early anatomical restoration of the macula . we believe that occult cnv and a cnv smaller than 5 da are optimistic for better morphological therapeutic response at the beginning of ranibizumab therapy . to determine if other factors influence the morphological response to ranibizumab treatment in patients with wet amd , further clinical studies are needed . few similar cases are reported in literature but none of them is associated with mirizzi syndrome . radiological images could help the surgeon to choose the surgical strategy and to evaluate the presence of associated diseases . in our case laparoscopic surgery showed to be an excellent approach for both the cholecystectomy and the repair of the defect of the abdominal wall . the patient was an 85 year - old man with history of hypertension , previous surgery for perforated diverticular disease ( hartmann procedure followed by reversal of colostomy ) . the physical examination showed a 5 cm mass in the right upper quadrant tender to palpation . ultrasound and ct confirmed the presence of a lithiasic gallbladder herniated through the abdominal wall ( fig.1 ) , with a dilatation of the common bile duct that measured 11 mm , due to a 17 mm obstructive infundibular stone as it happens in mirizzi syndrome type i ( fig.2 ) . lithiasic gallbladder herniated through the abdominal wall the routine complete blood test , including bilirubin level , was unremarkable . therefore the patient underwent a laparoscopic cholecystectomy and a repair of the hernia . during surgery , multiple adhesions were found and released carefully to identify the gallbladder that was fully included in the right upper quadrant abdominal wall covered by peritoneum . the gallbladder was completely released and the dissection of the pedicle showed a short cystic duct and a normal common bile duct . most of the cases reported in literature are internal hernias through the winslow foramen ( 1 ) . the herniation of the gallbladder through acquired defects of the abdominal wall ( incisional hernia ) has been much less reported . ( 2 ) presented the case of a gallbladder herniated through a fascial defect of a subcostal incision . garcia reported a patient who presented a gallbladder hernia through a parastomal defect ( 3 ) . more recently , the case of a gallbladder strangulation through an abdominal wall defect on the site of a previous colostomy was described ( 4 ) . in this case , the most remarkable thing is that herniation does not occur through a natural orifice or an acquired defect , such as respectively for the winslow foramen or for an incisional hernia , but directly into the abdominal wall . to our knowledge , there are only 3 published cases of spontaneous herniation of the gallbladder through the abdominal wall ( 5,6,7 ) . another interesting aspect of this case is the presence of a chronically distended gallbladder ( gallbladder hydrops ) , associated with extrinsic compression of the common hepatic duct by an impacted stone in the infundibulum ( mirizzi syndrome type i ) ( 8) . it is probably the gallbladder dilatation that plays an important role in the development of the hernia pressing constantly the gallbladder against the abdominal wall . usually the clinical picture is represented by a right upper quadrant pain , associated with variable degrees of a compromised general condition . in our experience the preoperative study based on computed tomography is essential for the diagnosis of the gallbladder hernia , and it provides also additional information on the abdominal wall defect . the management of this type of hernia consists in reducing the content and repairing the abdominal wall defect . in this case we suggest the laparoscopy as surgical approach of first choice because it allows to solve three problems at the same time : first , the reduction of the gallbladder s incarcerated hernia ; second , it enables to perform a cholecystectomy that was indicated for the presence of gallstones and hydrops ; third , it allows to repair the hernia defect preferably with a mesh , if the local conditions are favorable . in our experience , preoperative imaging has been proved to detect the gallbladder disease and the morphology of the fascial defect . mesh repair is the gold standard for hernias ; however , acute cholecystitis still remains a contraindication for mesh repair due to the high risk of infection .

aim . to assess the significance of age , gender , baseline best corrected visual acuity , baseline macula thickness , and type and size of choroidal neovascularization in early morphological therapeutic response to ranibizumab treatment in patients with the wet form of age - related macular degeneration . methods . from 09/2008 to 06/2013 we evaluated 1153 newly diagnosed , treatment - nave patients treated with ranibizumab . based on the morphological findings in the macula following the initial 3 injections of ranibizumab , the patients were divided into two groups based on active and inactive choroidal neovascularization . results . after the initial 3 injections of ranibizumab , we examined the sample of 841 eyes with active cnv and 312 eyes with inactive cnv . in the inactive group , we found a statistically higher proportion of occult cnv ( p < 0.001 ) and lower incidence of cnv greater than 5da ( p < 0.001 ) compared with the active group . we found no statistically significant difference in age , gender , baseline best corrected visual acuity , or baseline macula thickness between the inactive and active groups . conclusion . occult cnv and cnv smaller than 5da are optimistic factors for a better morphological therapeutic response at the beginning of ranibizumab treatment . a gallbladder incarcerated hernia associated with mirizzi syndrome is a very rare entity and to our knowledge this is the first case ever described in literature . an 85-year - old man presented at the emergency department with a tender right upper quadrant mass . computed tomography ( ct ) revealed the presence of a gallbladder lithiasis with signs of acute cholecystitis , herniated through the abdominal wall with an associated mirizzi syndrome . laparoscopic cholecystectomy and repair of the abdominal wall defect were performed . the patient recovered very well and the postoperative period was uneventful .

neglected tropical diseases ( ntds ) is an umbrella term used to describe infectious diseases that primarily affect low - income populations in tropical regions and are often widespread , chronic , and debilitating . although the number of diseases that are officially recognized as ntds by various public health authorities ranges from less than 10 to over 40 , the list of 17 diseases currently classified as ntds by the world health organization ( who ) is widely accepted as accurate and comprehensive ( 20 ) . of these 17 diseases , four are caused by bacterial pathogens ( buruli ulcer , leprosy , trachoma , and yaws ) , three are caused by protozoan parasites ( chagas disease , human african trypanosomiasis and leishmaniasis ) , two are viral ( dengue and rabies ) , and the other eight are caused by various species of parasitic worms ( 20 ) . the most recent data available indicating the estimated number of cases of each ntd are listed in table 1 . estimated number of cases of neglected tropical diseases ( ntds ) worldwide . prevalence ( total number of existing and new cases ) is reported for chronic ntds and incidence ( total number of new cases ) is reported for acute ntds or ntds with a high rate of recurrence . cdc , centers for disease control ; cfsph , center for food security and public health , iowa state university ; n / a , data not available ; who , world health organization . ntds exert an immensely deleterious effect on the public health and economies of much of the developing world . yet , by definition , ntds are issues for which the levels of awareness and resources necessary to develop effective solutions have not been sufficiently garnered . because ntds are largely confined to poor areas and have relatively low mortality rates , the policy makers and financiers of global health campaigns may underestimate or simply not be aware of the magnitude of the problem . it is for precisely these reasons ( that ntds are chronically debilitating , affect a large number of indigent people , and are often overlooked ) that it is crucial to increase the amount of education , research , and public outreach devoted to their study and treatment . even though ntds are largely confined to developing countries , there are a number of reasons why addressing ntds is important to developed countries such as the united states , and to the educational systems therein . the argument for increasing the focus on teaching about ntds can be distilled into three main points : ( i ) the sheer magnitude of the impact that ntds have on global health and interconnected world economies warrants a proportionately significant amount of attention in our classrooms ( ii ) increased awareness of ntds in western countries is crucial because successful ntd research programs and public health campaigns can significantly benefit from leadership , expertise , and funding from those with the greatest resources , and ( iii ) teaching and learning about ntds provides a unique opportunity to bridge myriad connections between basic science and other disciplines and to promote civic engagement and critical thinking . this is nearly 40 times the number of people living with hiv ( 37 million ) and more than six times the annual number of cases of malaria reported in recent years ( approximately 216 million ) ( 12 , 19 ) . although schistosomiasis alone is responsible for approximately 200,000 deaths per year , most ntds have relatively low mortality rates ( 20 ) . however , ntds can be permanently debilitating or disfiguring , and significantly limit the educational opportunities , productivity , and wage earning potential of affected individuals ( 16 ) . for example , trachoma , the leading cause of preventable blindness in the world , currently impairs the sight of over 8 million people and costs approximately $ 2.9 billion in lost productivity each year ( 7 , 16 ) . even more striking is the centers for disease control ( cdc ) estimate that the economic benefit of a successful campaign to eliminate lymphatic filariasis could exceed $ 55 billion ( 7 ) . as shown in table 1 , these two diseases are far from being the most widespread ntds , which underscores the immensity of this issue . the total impact of ntds is difficult to estimate due to a variety of factors including the chronic nature of these diseases , the confounding effects of multiple comorbidities , and difficulties in accurately tracking and treating diseases in large , diverse , poor populations . the effect of ntds is perhaps most accurately expressed as the amount of healthy productive years of life lost due to a combination of chronic disability and premature death of affected individuals . worldwide , ntds are responsible for the loss of approximately 4657 million years of healthy life per annum ( 16 ) . the only disease that currently causes a greater burden of this type is aids ( 16 ) . in addition to causing loss of human life and productivity , the pathogens that cause ntds further devastate poor communities by harming the livestock on which subsistence farmers depend for their livelihood . african trypanosomes , closely related to those that cause human african trypanosomiasis , are responsible for a widespread and devastating disease in livestock called nagana , which kills three million cattle per year , amounting to a loss of $ 4 billion from struggling african economies . similarly , cysticercosis causes a substantial economic toll by reducing the market value of cattle and pigs , which are rendered unsafe to eat when infected ( 20 ) . because we live in an increasingly interconnected world , the economic and social ramifications of ntds are not confined to endemic nations ; when the health and productivity of one - fifth of the world s population is adversely affected by disease , other nations are indirectly impacted by the moral obligation to provide assistance and the cost of providing that aid . more than 300,000 individuals currently residing in the united states are infected with trypanosoma cruzi , the causative agent of chagas disease , for which there is no vaccine available ( 7 ) . although most of these cases are attributed to infections that occurred outside of the country , at least 31 people have been infected inside the continental united states via vectoral transmission or transfusion of infected blood products ( 5 , 13 ) . and , as recently as 2008 , soil - transmitted helminth infections in the united states were estimated at nearly 4 million , occurring mostly in poor regions of appalachia and the south ( 10 ) . even ntds of which there are few , if any , domestic human cases reported can still exert a great impact on a country . for example , over $ 300 million is spent each year in the united states alone to vaccinate animals against rabies in order to prevent human infection ( 7 ) . the incongruous amount of attention devoted to ntds compared to other less prevalent diseases , such as aids , malaria , and tuberculosis ( often referred to as the big three diseases ) , extends beyond the allocation of funding for scientific research and public health campaigns and into the arena of public education . peter hotez , dean of the national school of tropical medicine at baylor college of medicine , has described ntds as the most common diseases you have never heard of , a characterization that should be actively changed through modifications to our nation s education system ( 9 ) . the typical american student enrolled in a general microbiology course is likely to know and learn significantly less about ntds than hiv / aids , even though the incidence of ntds is much higher . for example , the textbook chosen by this author s department for use in all microbiology courses , which is a highly regarded text described by the publisher , john wiley & sons , inc . , as a bestseller for multiple editions , discusses ntds on less than 10% of approximately 200 pages devoted to describing infectious diseases . although the prevalence of a disease is only one of many factors to consider when allocating class time to discussing various pathogens , the striking epidemiology of ntds , along with the other factors discussed herein , make a strong case for devoting a significant amount of time to the study of these diseases . the educational merits of teaching about ntds and the effects of these diseases on american citizens are not the only reasons to teach our students about them . the united states plays a crucial role in the global fight against ntds , yet our efforts as a nation have been historically inadequate ; as of 2012 , only 1.3% of the money spent by the united states on global health programs was devoted to ntds ( 16 ) . in contrast , malaria and hiv / aids ( which are much less prevalent ) account for 60% of the nation s global health budget ( 16 ) . non - governmental foundations based in the united states , such as the gates foundation , also provide a significant portion of the global budget for combating ntds ; however , there is widespread pressure to tighten budgets and decrease funding of global health programs in both the public and private sector . equally important to the funding of public health initiatives is the expertise of american scientists and physicians in developing chemotherapeutics that target the causative agents of ntds , and funding for basic science research has been drastically cut in recent years . our efforts must be expanded , not stymied , in order to make progress in eradicating ntds . increasing awareness of ntds through education , and promoting the interest of our nation s students in becoming ntd researchers and health policy makers , is likely the most promising way to ensure the continuation and redoubling of our nation s efforts to combat these diseases . due to the tremendously far - reaching and long - lasting impact that ntds exert on the human race , teaching about these diseases provides a prime opportunity to incorporate interdisciplinary learning and civic engagement into science education . the benefits of interdisciplinary learning , which involves the integration of knowledge from multiple fields of study around a central theme , include helping students access prior knowledge in order to form connections with new knowledge , the promotion of critical thinking and higher - order cognitive skills , improvement of content retention , enhancement of creativity , increased motivation to learn , and increased sensitivity to ethical issues ( 11 , 14 ) . microbiology lessons about ntds could easily be paired with an array of courses to facilitate interdisciplinary learning by emphasizing the link between the biology of ntds and socioeconomic issues . pairing a microbiology course with a public health course could facilitate interdisciplinary learning by focusing on the development of improved health policies and practices geared towards ameliorating the effects of ntds in developing nations . collaboration with epidemiology or mathematics courses could focus on quantifying the impact of ntds , and utilize real - world ntd - related problems as examples for teaching mathematical concepts . an interdisciplinary approach involving chemistry or pharmacology courses could focus on the development of improved chemotherapeutics to treat ntds , which is an area of global medicine significantly lacking in funding and progress . strategies for mobilizing and managing resources to treat and eliminate ntds and to sever the connection between ntds and the propagation of poverty could be explored in business , economics , or public policy courses . because the influence of ntds on the health and economies of endemic countries has been so forceful and enduring as to shape the histories of those countries , african studies , asian studies , and latin american studies programs the broad implications of ntds could be further explored in sociology courses by examining the way these diseases have shaped societies over time , and the ethical implications of how those with the resources to make a difference respond to the plight of those in need . all of the aforementioned issues could be further explored by integrating a writing - intensive course in which students can thoroughly develop and communicate their understanding and reactions . civic engagement , or the application of knowledge and skills into actions that promote and improve the well being of communities , is becoming increasingly emphasized as an important part of high school and university curricula . as with interdisciplinary learning , the extensive impact ntds have on communities around the world makes teaching about ntds well suited to the incorporation of civic engagement . the thought of civic engagement related to ntds may conjure images of students conducting fieldwork in remote tropical locations , but that is only one way to incorporate civic engagement in the study of ntds . because ntds leave almost no one in the world completely unaffected due to the potential for infection in non - endemic regions , the indirect economic impact that ntds exert on essentially every nation , and the moral obligation of those with the resources to help , any project that increases awareness of these issues in any community could be a valid civic engagement endeavor . interdisciplinary learning and civic engagement promote active learning by engaging students in critical thinking , reflective reasoning , data analysis , assessing the validity of data sources , synthesizing ideas and solutions , and interpreting knowledge with cultural literacy . active learning , in turn , helps propel students through the levels of bloom s taxonomy of learning ( 2 ) , from knowledge acquisition to applying , analyzing , and evaluating scientific information to synthesize solutions to problems . there are a number of pedagogical tools available to facilitate teaching and learning about ntds . in addition to the material about ntds presented in textbooks , an array of materials that can be used to supplement instruction is freely available . the global health division of the cdc maintains an easily navigable encyclopedic resource about ntds on its website ( http://www.cdc.gov/globalhealth/ntd/ ) , providing information about the biology , disease signs and symptoms , diagnostic and treatment options , epidemiology and risk factors , and prevention and control measures for each disease . the scope and presentation of this material makes it a felicitous resource for engaging students in research and active learning . the who provides a similar collection of information about ntds on its website ( http://www.who.int/neglected_diseases/diseases/en/ ) , including a cache of interesting facts about each ntd that are not all easily found on the cdc website ; however , the organization and presentation of information by the who is less accessible than on the cdc website , making it better suited as a secondary , complementary resource . additional resources include : alliance for case studies for global health ( http://www.casestudiesforglobalhealth.org ) , global network for neglected tropical diseases ( http://www.globalnetwork.org ) , pan american health organization ( http://new.paho.org ) , end neglected tropical diseases in africa ( end in africa ) ( http://endinafrica.org/ ) , the neglected tropical disease nongovernmental development organizations network ( http://ntd-ngdonetwork.org/ ) , and the united states agency for international development ntd program ( http://www.neglecteddiseases.gov/ ) . these websites may be useful for promoting interdisciplinary discussion and critical thinking because they provide a wealth of information about current and planned public health initiatives , as well as facts , anecdotes , and photographs that illustrate the human toll and vast socioeconomic implications of these diseases . some courses may benefit from the analysis and discussion of primary literature , a plethora of which is available via national center for biotechnology information ( 15 ) . the scholarly journal plos neglected tropical diseases is devoted exclusively to ntds , and many other scientific journals regularly publish primary research , review articles , and commentaries about ntds . the national center for case study teaching in science ( nccsts ) at the university of buffalo maintains a database of peer - viewed case studies that are freely available for use in the classroom . the nccsts database is continually being expanded , and currently includes at least five case studies about ntds , one on african trypanosomiasis ( 3 ) , two about chagas disease ( 4 , 18 ) and two on dengue fever ( 1 , 17 ) . these cases are presented as multipart narratives interrupted by questions and active learning exercises . for instructor use only , a password - protected answer key is provided , along with a set of teaching notes describing how the authors implemented the case in their own classroom and tips they have for other instructors who wish to use the case . many pedagogical tools are utilized in the case studies , including small and large group discussions , brainstorming , problem - based learning , and group presentations that incorporate a research component and peer feedback . to complete the case studies , students analyze scientific data and contextual information to identify a particular ntd - causing pathogen , and evaluate existing public health campaigns in order to synthesize their own plan for effectively treating and preventing the spread of that disease . these case studies provide a framework for examining the interdisciplinary connection of microbiology with fields such as economics , epidemiology , public health , and sociology , and promote civic engagement by emphasizing the connection between biology and the complex real - world problems facing communities affected by ntds . to promote hands - on civic engagement , there are a number of established programs available for educators who have the opportunity to incorporate a study - abroad component into their curriculum . two programs specifically designed to engage undergraduate students in laboratory and field - based research and community outreach projects in areas that are endemic for ntds are the public health brigades , overseen by the global brigades association headquartered in seattle , washington , and the tropical disease research program directed by dr . both of these programs are open to qualified students at colleges around the country and are primarily associated with public health projects related to chagas disease . the public health brigades program involves a week - long excursion in rural honduras , where students directly engage with locals in home infrastructural development , community leader training , and health education programs ( 8) . the tropical disease research program engages students in hands - on modules involving field studies , laboratory research , and healthy living development projects in rural ecuador over a period of two to six weeks ( 19 ) . many other opportunities for qualified students to conduct research and fieldwork exist , but due to variability in the availability and specifications of these programs they will not be described here . to reiterate , relevant civic engagement projects need not involve a travel - abroad component as long as they increase the awareness of the risks , social issues , or need for funding associated with ntds . this is nearly 40 times the number of people living with hiv ( 37 million ) and more than six times the annual number of cases of malaria reported in recent years ( approximately 216 million ) ( 12 , 19 ) . although schistosomiasis alone is responsible for approximately 200,000 deaths per year , most ntds have relatively low mortality rates ( 20 ) . however , ntds can be permanently debilitating or disfiguring , and significantly limit the educational opportunities , productivity , and wage earning potential of affected individuals ( 16 ) . for example , trachoma , the leading cause of preventable blindness in the world , currently impairs the sight of over 8 million people and costs approximately $ 2.9 billion in lost productivity each year ( 7 , 16 ) . even more striking is the centers for disease control ( cdc ) estimate that the economic benefit of a successful campaign to eliminate lymphatic filariasis could exceed $ 55 billion ( 7 ) . as shown in table 1 , these two diseases are far from being the most widespread ntds , which underscores the immensity of this issue . the total impact of ntds is difficult to estimate due to a variety of factors including the chronic nature of these diseases , the confounding effects of multiple comorbidities , and difficulties in accurately tracking and treating diseases in large , diverse , poor populations . the effect of ntds is perhaps most accurately expressed as the amount of healthy productive years of life lost due to a combination of chronic disability and premature death of affected individuals . worldwide , ntds are responsible for the loss of approximately 4657 million years of healthy life per annum ( 16 ) . the only disease that currently causes a greater burden of this type is aids ( 16 ) . in addition to causing loss of human life and productivity , the pathogens that cause ntds further devastate poor communities by harming the livestock on which subsistence farmers depend for their livelihood . african trypanosomes , closely related to those that cause human african trypanosomiasis , are responsible for a widespread and devastating disease in livestock called nagana , which kills three million cattle per year , amounting to a loss of $ 4 billion from struggling african economies . similarly , cysticercosis causes a substantial economic toll by reducing the market value of cattle and pigs , which are rendered unsafe to eat when infected ( 20 ) . because we live in an increasingly interconnected world , the economic and social ramifications of ntds are not confined to endemic nations ; when the health and productivity of one - fifth of the world s population is adversely affected by disease , other nations are indirectly impacted by the moral obligation to provide assistance and the cost of providing that aid . more than 300,000 individuals currently residing in the united states are infected with trypanosoma cruzi , the causative agent of chagas disease , for which there is no vaccine available ( 7 ) . although most of these cases are attributed to infections that occurred outside of the country , at least 31 people have been infected inside the continental united states via vectoral transmission or transfusion of infected blood products ( 5 , 13 ) . and , as recently as 2008 , soil - transmitted helminth infections in the united states were estimated at nearly 4 million , occurring mostly in poor regions of appalachia and the south ( 10 ) . even ntds of which there are few , if any , domestic human cases reported can still exert a great impact on a country . for example , over $ 300 million is spent each year in the united states alone to vaccinate animals against rabies in order to prevent human infection ( 7 ) . the incongruous amount of attention devoted to ntds compared to other less prevalent diseases , such as aids , malaria , and tuberculosis ( often referred to as the big three diseases ) , extends beyond the allocation of funding for scientific research and public health campaigns and into the arena of public education . peter hotez , dean of the national school of tropical medicine at baylor college of medicine , has described ntds as the most common diseases you have never heard of , a characterization that should be actively changed through modifications to our nation s education system ( 9 ) . the typical american student enrolled in a general microbiology course is likely to know and learn significantly less about ntds than hiv / aids , even though the incidence of ntds is much higher . for example , the textbook chosen by this author s department for use in all microbiology courses , which is a highly regarded text described by the publisher , john wiley & sons , inc . , as a bestseller for multiple editions , discusses ntds on less than 10% of approximately 200 pages devoted to describing infectious diseases . although the prevalence of a disease is only one of many factors to consider when allocating class time to discussing various pathogens , the striking epidemiology of ntds , along with the other factors discussed herein , make a strong case for devoting a significant amount of time to the study of these diseases . the educational merits of teaching about ntds and the effects of these diseases on american citizens are not the only reasons to teach our students about them . the united states plays a crucial role in the global fight against ntds , yet our efforts as a nation have been historically inadequate ; as of 2012 , only 1.3% of the money spent by the united states on global health programs was devoted to ntds ( 16 ) . in contrast , malaria and hiv / aids ( which are much less prevalent ) account for 60% of the nation s global health budget ( 16 ) . non - governmental foundations based in the united states , such as the gates foundation , also provide a significant portion of the global budget for combating ntds ; however , there is widespread pressure to tighten budgets and decrease funding of global health programs in both the public and private sector . equally important to the funding of public health initiatives is the expertise of american scientists and physicians in developing chemotherapeutics that target the causative agents of ntds , and funding for basic science research has been drastically cut in recent years . our efforts must be expanded , not stymied , in order to make progress in eradicating ntds . increasing awareness of ntds through education , and promoting the interest of our nation s students in becoming ntd researchers and health policy makers , is likely the most promising way to ensure the continuation and redoubling of our nation s efforts to combat these diseases . due to the tremendously far - reaching and long - lasting impact that ntds exert on the human race , teaching about these diseases provides a prime opportunity to incorporate interdisciplinary learning and civic engagement into science education . the benefits of interdisciplinary learning , which involves the integration of knowledge from multiple fields of study around a central theme , include helping students access prior knowledge in order to form connections with new knowledge , the promotion of critical thinking and higher - order cognitive skills , improvement of content retention , enhancement of creativity , increased motivation to learn , and increased sensitivity to ethical issues ( 11 , 14 ) . microbiology lessons about ntds could easily be paired with an array of courses to facilitate interdisciplinary learning by emphasizing the link between the biology of ntds and socioeconomic issues . pairing a microbiology course with a public health course could facilitate interdisciplinary learning by focusing on the development of improved health policies and practices geared towards ameliorating the effects of ntds in developing nations . collaboration with epidemiology or mathematics courses could focus on quantifying the impact of ntds , and utilize real - world ntd - related problems as examples for teaching mathematical concepts . an interdisciplinary approach involving chemistry or pharmacology courses could focus on the development of improved chemotherapeutics to treat ntds , which is an area of global medicine significantly lacking in funding and progress . strategies for mobilizing and managing resources to treat and eliminate ntds and to sever the connection between ntds and the propagation of poverty could be explored in business , economics , or public policy courses . because the influence of ntds on the health and economies of endemic countries has been so forceful and enduring as to shape the histories of those countries , african studies , asian studies , and latin american studies programs the broad implications of ntds could be further explored in sociology courses by examining the way these diseases have shaped societies over time , and the ethical implications of how those with the resources to make a difference respond to the plight of those in need . all of the aforementioned issues could be further explored by integrating a writing - intensive course in which students can thoroughly develop and communicate their understanding and reactions . civic engagement , or the application of knowledge and skills into actions that promote and improve the well being of communities , is becoming increasingly emphasized as an important part of high school and university curricula . as with interdisciplinary learning , the extensive impact ntds have on communities around the world makes teaching about ntds well suited to the incorporation of civic engagement . the thought of civic engagement related to ntds may conjure images of students conducting fieldwork in remote tropical locations , but that is only one way to incorporate civic engagement in the study of ntds . because ntds leave almost no one in the world completely unaffected due to the potential for infection in non - endemic regions , the indirect economic impact that ntds exert on essentially every nation , and the moral obligation of those with the resources to help , any project that increases awareness of these issues in any community could be a valid civic engagement endeavor . interdisciplinary learning and civic engagement promote active learning by engaging students in critical thinking , reflective reasoning , data analysis , assessing the validity of data sources , synthesizing ideas and solutions , and interpreting knowledge with cultural literacy . active learning , in turn , helps propel students through the levels of bloom s taxonomy of learning ( 2 ) , from knowledge acquisition to applying , analyzing , and evaluating scientific information to synthesize solutions to problems . there are a number of pedagogical tools available to facilitate teaching and learning about ntds . in addition to the material about ntds presented in textbooks , an array of materials that can be used to supplement instruction is freely available . the global health division of the cdc maintains an easily navigable encyclopedic resource about ntds on its website ( http://www.cdc.gov/globalhealth/ntd/ ) , providing information about the biology , disease signs and symptoms , diagnostic and treatment options , epidemiology and risk factors , and prevention and control measures for each disease . the scope and presentation of this material makes it a felicitous resource for engaging students in research and active learning . the who provides a similar collection of information about ntds on its website ( http://www.who.int/neglected_diseases/diseases/en/ ) , including a cache of interesting facts about each ntd that are not all easily found on the cdc website ; however , the organization and presentation of information by the who is less accessible than on the cdc website , making it better suited as a secondary , complementary resource . additional resources include : alliance for case studies for global health ( http://www.casestudiesforglobalhealth.org ) , global network for neglected tropical diseases ( http://www.globalnetwork.org ) , pan american health organization ( http://new.paho.org ) , end neglected tropical diseases in africa ( end in africa ) ( http://endinafrica.org/ ) , the neglected tropical disease nongovernmental development organizations network ( http://ntd-ngdonetwork.org/ ) , and the united states agency for international development ntd program ( http://www.neglecteddiseases.gov/ ) . these websites may be useful for promoting interdisciplinary discussion and critical thinking because they provide a wealth of information about current and planned public health initiatives , as well as facts , anecdotes , and photographs that illustrate the human toll and vast socioeconomic implications of these diseases . some courses may benefit from the analysis and discussion of primary literature , a plethora of which is available via national center for biotechnology information ( 15 ) . the scholarly journal plos neglected tropical diseases is devoted exclusively to ntds , and many other scientific journals regularly publish primary research , review articles , and commentaries about ntds . the national center for case study teaching in science ( nccsts ) at the university of buffalo maintains a database of peer - viewed case studies that are freely available for use in the classroom . the nccsts database is continually being expanded , and currently includes at least five case studies about ntds , one on african trypanosomiasis ( 3 ) , two about chagas disease ( 4 , 18 ) and two on dengue fever ( 1 , 17 ) . these cases are presented as multipart narratives interrupted by questions and active learning exercises . for instructor use only , a password - protected answer key is provided , along with a set of teaching notes describing how the authors implemented the case in their own classroom and tips they have for other instructors who wish to use the case . many pedagogical tools are utilized in the case studies , including small and large group discussions , brainstorming , problem - based learning , and group presentations that incorporate a research component and peer feedback . to complete the case studies , students analyze scientific data and contextual information to identify a particular ntd - causing pathogen , and evaluate existing public health campaigns in order to synthesize their own plan for effectively treating and preventing the spread of that disease . these case studies provide a framework for examining the interdisciplinary connection of microbiology with fields such as economics , epidemiology , public health , and sociology , and promote civic engagement by emphasizing the connection between biology and the complex real - world problems facing communities affected by ntds . to promote hands - on civic engagement , there are a number of established programs available for educators who have the opportunity to incorporate a study - abroad component into their curriculum . two programs specifically designed to engage undergraduate students in laboratory and field - based research and community outreach projects in areas that are endemic for ntds are the public health brigades , overseen by the global brigades association headquartered in seattle , washington , and the tropical disease research program directed by dr . both of these programs are open to qualified students at colleges around the country and are primarily associated with public health projects related to chagas disease . the public health brigades program involves a week - long excursion in rural honduras , where students directly engage with locals in home infrastructural development , community leader training , and health education programs ( 8) . the tropical disease research program engages students in hands - on modules involving field studies , laboratory research , and healthy living development projects in rural ecuador over a period of two to six weeks ( 19 ) . many other opportunities for qualified students to conduct research and fieldwork exist , but due to variability in the availability and specifications of these programs they will not be described here . to reiterate , relevant civic engagement projects need not involve a travel - abroad component as long as they increase the awareness of the risks , social issues , or need for funding associated with ntds . ntds are often marginalized in the spheres of global health policy and scientific research , as well as in the classroom . there is a strong argument for restructuring microbiology curriculum to increase the emphasis on teaching and learning about ntds due to the fact that these diseases affect more people worldwide than almost any other group of diseases and because educating students about the seriousness of ntds is integral to ensuring that the future leaders of the world will focus sufficient resources on ameliorating the global plight caused by this group of diseases . ntds also provide an opportunity to teach and learn about a diverse and interesting group of organisms , while promoting interdisciplinary learning , civic engagement , and critical thinking . to promote the teaching of ntds , educators have an array of educational tools to choose from , including informational websites , a case study database , and inter - institutional study - abroad programs . severely obese adults seeking lifestyle interventions report impaired physical , mental , and obesity - specific quality of life ( qol ) [ 13 ] . thus , several studies have included qol as a primary outcome in evaluation of multicomponent lifestyle interventions for these individuals [ 47 ] . these studies have proposed physical activity to be a contributor to unexplained improvements in qol [ 47 ] . . demonstrated improvements in the physical component summary ( pcs ) score and mental component summary ( mcs ) score of the medical outcomes study short - form 36 health survey ( sf-36 ) at the end of one - year , partly residential interventions . reported similar findings after a six - month , outpatient intervention for overweight to obese adults . repeated measures in two of these studies revealed improvements in both pcs and mcs after an initial , intensive intervention phase whereas longer - term maintenance varied [ 4 , 7 ] . the results reported by blissmer et al . . revealed positive associations between weight loss and improvements in pcs but not mcs . with regard to obesity - specific qol , the swedish obese subjects ( sos ) study developed and used the instrument obesity - related problems ( op ) scale . after four and ten years , the authors found significant improvements in severely obese adults who had received conventional treatment . however , the treatment was not standardised but was provided in accordance with local routines by many primary health care centres . in contrast , despite weight regain , kaukua et al . demonstrated improvements in op at the end of a two - year follow - up of severely obese individuals who completed a four - month , outpatient intervention . compared to physical , mental , and obesity - specific qol , life satisfaction is a broader qol construct representing a subjective and global assessment of all major dimensions of life . using single item measures on life satisfaction , obesity was associated with impaired life satisfaction in two u.s . population studies [ 10 , 11 ] , whereas a danish epidemiological study , controlling for a cluster of lifestyle - related factors including body mass index ( bmi ) , found independent positive associations between self - reported physical activity and life satisfaction . multicomponent lifestyle interventions for severe obesity aim for a sustainable change of behaviour related to diet and physical activity . physical activity is beneficial for body composition and fitness in obese individuals undergoing dietary energy restriction and reduces adverse cardiovascular outcomes of obesity [ 15 , 16 ] . in the eight - year follow - up of the look ahead study , self - reported physical activity was associated with initial and maintained weight loss in overweight to obese subjects with type 2 diabetes . cross - sectional , unadjusted analyses in studies on treatment - seeking severely obese adults have demonstrated positive correlations between self - reported physical activity and pcs [ 18 , 19 ] and mcs . we found positive independent associations between objectively assessed physical activity and life satisfaction prior to a lifestyle intervention for severely obese adults and did a series of studies on patterns of change in the participants during the intervention . we used accelerometers to objectively measure physical activity , collected data at multiple time points , and found positive associations in the patterns of change between physical activity and aerobic fitness , fat mass , and lipoproteins , confirming the importance of physical activity for clinical and anthropometric outcomes in lifestyle interventions . to our knowledge , a similar design has not been used to examine associations between change in physical activity and qol outcomes over time . therefore , the present study examines associations between change in objectively assessed physical activity as the independent variable and change in physical , mental , and obesity - specific qol and life satisfaction as the dependent variables during a two - year , multicomponent lifestyle intervention . this study is part of the haugland obesity study , a prospective cohort study on severely obese adults who participated in a publicly funded two - year lifestyle intervention at red cross haugland rehabilitation centre ( rchrc ) in western norway . data were collected between february 2010 and october 2012 and the present study used data from four time points : baseline prior to the intervention ( t0 ) , six weeks later , at the end of the first residential stay , ( t1 ) , and prior to the residential stays one ( t2 ) and two ( t3 ) years from baseline . referral of patients was done by general practitioners in accordance with the right to admission to the norwegian specialist health services ( i.e. , bmi 40 kg / m or 35 kg / m with comorbidities ) . in total , 53 eligible patients from the age of 18 to 60 years , divided into four groups , had their first residential stay between february 2010 and october 2011 . exclusion criteria included previous obesity surgery or referral to obesity surgery ; severe cardiovascular disease ; pregnancy ; substance or alcohol abuse ; and impaired physical functioning or mental problems which could interfere with adherence to the intervention . briefly , the patients spent a total of 15 weeks at rchrc divided into four stays of six , three ( after three months ) , three ( at year one ) , and three ( at year two ) weeks . the overall goal was to improve the qol of the patients , while weight loss , improved mental health and physical fitness , and reduction of obesity - related medical problems served as secondary goals . the group - based cognitive behavioural therapy , consisting of eleven sessions over two years , targeted qol and self - management of physical activity and eating . scheduled physical activity in the residential periods consisted of brisk walking , swimming , strength training , ball games , and aerobics and amounted to nine to eleven hours weekly divided into bouts of 2060 minutes . each patient developed a plan for physical activity , modified to his or her preferences , limitations , and home situation . the diet followed the nordic nutrition recommendations and consisted of three high - fibre , low - fat , and energy - reduced meals and two to three snacks . the patients were advised to follow a similar diet at home . in the home periods , pcs ranges from 15.4 to 62.1 and mcs from 10.1 to 64.0 ( with higher scores representing better qol ) [ 28 , 29 ] . the pcs and mcs have been standardised to a population normal distribution , with a mean of 50 and a standard deviation ( sd ) of 10 . sf-36 has been widely applied in obesity research [ 1 , 30 ] , discriminates between subgroups of severely obese adults , and is sensitive to change during lifestyle interventions [ 4 , 5 ] . this is an eight - item measure of obesity - specific qol including questions about restaurant visits , holidaying , participation in community activities , swimming in public places , trying on and buying clothes , and intimate / sexual situations . the calibrated score ranges from 0 to 100 ( < 40 mild , 40 to < 60 moderate , 60 to < 80 severe , and 80 extreme problems ) . op is reliable and valid in severely obese adults [ 2 , 31 ] . in the present study , life satisfaction was assessed using a global question on current satisfaction with life with seven response alternatives from very satisfied to very dissatisfied . one - item measures on life satisfaction have demonstrated reliability and validity in health research [ 33 , 34 ] . to assess physical activity , we used the actigraph gti m accelerometer ( actigraph , pensacola , fl , usa ) , which is an electronic movement sensor . the accelerometer registers vertical acceleration and converts it into the unit counts which increase with the magnitude of the work rate for walking . the participants were instructed to wear the accelerometer over the right hip for seven consecutive days while awake , except during water activities . the t0 , t2 , and t3 assessments took place during home periods , while the t1 assessment was carried out at the end of the first residential period . the criterion for a valid measure was wear - time of ten hours per day for four days . non - wear - time was defined as periods of 60 consecutive minutes without counts , allowing for up to two minutes of counts within these 60 minutes [ 35 , 36 ] . the overall physical activity , given as counts per minute , was calculated as total counts divided by total valid wear - time . the accelerometer has shown validity in severely obese individuals and accelerometer assessed physical activity offers more accuracy than self - reported data . height was measured in the standing position without shoes using a stadiometer and reported to the nearest 0.5 cm . fat mass and weight were measured on a bioelectrical impedance analysis device ( bc 420s ma , tanita corp . , tokyo , japan ) in the morning , in a fasting state , in light clothes , and after voiding . waist circumference was measured twice at exhalation at the level of the umbilicus and reported as the mean of the two measurements . we obtained written , informed consent from all participants prior to the study in accordance with the helsinki declaration . ethical approval was given by the regional committee for medical and health research ethics for south - east norway ( registration number 2010/159 ) . the scores on life satisfaction were reversed before analyses so that higher scores indicated better satisfaction with life . subject characteristics are presented as means and sd for continuous variables and percentages for categorical data . observed values for qol measures are presented as means and sd . the effect size ( es ) for differences between pcs and msc population norms and the study population scores at the four time points were calculated by subtracting the norms from the mean score of the participants divided by the sd of the latter . we performed an attrition analysis using the chi - squared test for difference in gender and the independent samples t - test for differences in other variables . a linear mixed model based on restricted maximum likelihood estimation with random intercept for subjects was used in all analyses for change ( ) over time , using least significant difference from baseline . the associations between the independent and the dependent variables were analysed using linear regression , applying delta scores between each time point ( y1 = y1 y0 ; x1 = x1 x0 ; y2 = y2 y1 , etc . ) , giving a total of n = 73 ( pcs and mcs ) , 72 ( op ) , and 71 ( life satisfaction ) observations . for physical activity , pcs , mcs , op , life satisfaction , and bmi , the differences between t0 and t1 ( 1 ) , t1 and t2 ( 2 ) , and t2 and t3 ( 3 ) were used . gender , age , and change in bmi served as covariates in the adjusted regression analyses . a 1000-repetition bootstrap analysis was used to calculate 95% confidence intervals ( ci ) of the regression coefficients . the changes from baseline to each of the time points in qol measures , physical activity , and bmi , obtained from the linear mixed model , are presented as means with 95% ci . the es for changes in the dependent variables were calculated by subtracting the mean t1 , t2 , and t3 estimates from the mean t0 estimate , divided by the sd of t0 . a secondary analysis was performed using a baseline - observation - carried - forward approach for missing values . effect sizes were judged against the standard criteria proposed by cohen : trivial ( < 0.2 ) , small ( 0.2 to < 0.5 ) , moderate ( 0.5 to < 0.8 ) , and large ( 0.8 ) . statistical analyses were conducted using spss for windows , version 20.0 ( spss inc . , chicago , usa ) . a two - sided p value of 0.05 indicated statistical significance . forty - nine patients ( 37 women ; 43.6 9.4 years ; bmi 42.1 6.0 kg / m ) consented to participate in the study . baseline sociodemographic and anthropometric characteristics are presented in table 1 . at year two , 16 women and six men ( 44.9% ) were lost to follow - up ( figure 1 ) . the rest dropped out of the intervention itself , due to referral to obesity surgery , pregnancy , reaching personal weight goals , health problems , inability to attend the residential stays , or unknown reasons . the noncompleters did not differ from the completers with regard to gender , age , bmi , physical activity , qol measures , or changes from t0 to t1 in bmi , physical activity , or qol measures . table 2 documents changes in the qol measures and the related es . over the first six weeks , , mcs and life satisfaction had returned to baseline levels , whereas the improvement in pcs was partly maintained . the es for within - group change was small for op , moderate for pcs and mcs , and large for life satisfaction after six weeks . at year one , the es was small for op and moderate for pcs . finally , at year two , the es were small for pcs and moderate for op . physical activity increased significantly during the first residential stay and was partly maintained at year one . at year two scores on pcs , mcs , op , and life satisfaction are presented in table 4 including es for differences between the study population and the norwegian sf-36 population norm . for pcs , figure 2 illustrates that correlations between change in physical activity and change in qol measures were strongest for mcs and weakest for op . this is also demonstrated in table 5 which presents the results of the regression analyses . in the adjusted analyses , changes in pcs , mcs , op , and life satisfaction the explained variance was moderate for pcs and mcs and small for op and life satisfaction . change in bmi was correlated with change in pcs , mcs , and life satisfaction in the unadjusted analyses ; however , this association was not statistically significant in the full models . replacement of change in bmi with change in waist circumference or fat mass did not alter any results ( data not shown ) . we tested for the interaction between physical activity and gender in all four models and found that the women had a stronger association between change in physical activity and change in pcs than the men ( p = 0.012 ) . this study has 71 to 73 observations for the main outcomes ( table 5 ) . given 71 observations , a power of 0.80 , and significance level of 0.05 , the study should have power to detect a standardized coefficient of 0.32 ( medium effect size ) . results from this study suggest that change in physical activity is independently associated with change in physical , mental , and obesity - specific qol and life satisfaction in severely obese adults participating in a lifestyle intervention . in correspondence with the present study , an independent dose - response relationship between exercise and physical and mental sf-36 subscales was reported from a randomized , controlled pre - post study on a six - month intervention for overweight to obese , menopausal , and hypertensive women . a cross - sectional study on overweight to obese subjects with type 2 diabetes also reported associations between self - reported physical activity and mcs , but not pcs , independent of bmi . by contrast , ross et al . reported that physical fitness , which may be a proxy for physical activity , did not mediate the association between weight reduction and improvements in sf-36 subscales in obese women enrolled in a six - month intervention . however , the six - minute walk test , which was utilized to assess fitness in the study by ross et al . used self - reported data on physical activity from two time points and found associations with pcs , but not mcs , in obesity surgery - seekers , although these results did not control for bmi . described improvements in op alongside fluctuations in self - reported physical activity during a lifestyle intervention but did not examine correlations . regarding life satisfaction , our cross - sectional baseline study from the haugland obesity study found an independent association with physical activity . population data revealed a positive relationship with self - reported physical activity , though this relationship did not control for bmi . a comparison of lifestyle interventions found no difference in life satisfaction between intervention and control groups during a one - year follow - up . so some studies do not support our finding of associations between change in physical activity and change in all qol outcomes . one explanation may be that the intervention of the present study clearly differed from other lifestyle interventions in that the overall goal was improvement of qol . other interventions have weight management [ 2 , 7 ] or behaviour change related to physical activity and diet ( which should lead to weight loss ) [ 46 ] as primary goals . the inconsistencies across studies may also relate to the cross - sectional design of several of them [ 10 , 20 , 43 ] , problems with the reliability of self - reported physical activity , the variety of weight classes included in the studies , and other heterogeneities of participants , context , or research designs . interestingly , although the unadjusted analyses revealed correlations between changes in bmi and pcs , mcs , and life satisfaction , weight loss did not moderate the associations between the independent and the dependent variables in the adjusted analyses . associations between weight loss and improvements in pcs have been found in several studies on patients undergoing lifestyle interventions [ 4 , 5 , 37 ] but not in the study by blissmer et al . . neither did kolotkin et al . after obesity surgery , whereas karlsen et al . our finding on op , as the only variable which had a nonsignificant correlation with change in bmi in the unadjusted analyses , is contradictive to the sos study which demonstrated short- and long - term decreases in obesity - related problems associated with weight loss [ 2 , 8 ] . noticeably , with regard to the purpose of the present study , none of the above - mentioned studies included physical activity as a variable . after an initial , in - patient period and kaukua et al . at the end of a four - month outpatient programme using the rand-36 questionnaire , equivalent to the sf-36 , and the op however , despite the statistically significant effect of physical activity on qol measures in our analyses , many of the improvements in the dependent variables were left unexplained . thus , several other aspects may have played a role , such as experience of peer support [ 46 , 47 ] , reduction of anxiety , improved eating pattern , and improved self - regulation and self - efficacy which have been demonstrated by others after intensive intervention phases , as well as our explicit intervention focus on improvement of qol . regarding the patterns of longer - term changes , there are variations across studies . our finding that mcs had returned to baseline at year two while moderate es for the change in pcs was maintained is opposite to two - year changes reported by blissmer el al . on overweight to obese subjects . the es for one - year changes reported for lifestyle intervention completers by karlsen et al . is similar to the es of the present study for pcs ( = 0.47 ) , but higher ( = 0.32 ) for mcs . yet the baseline scores on pcs ( mean = 39 , sd = 10 ) and mcs ( mean = 42 , sd = 11 ) were lower compared to our study which may indicate a greater potential for long - term improvements . so , although lifestyle treatment - seekers generally report better qol than obesity surgery - seekers and worse than non - treatment - seekers [ 1 , 3 ] , variations across study populations in lifestyle interventions may contribute to disparities in research outcomes . moreover , it may be unrealistic to expect the initial peaks in pcs , mcs , and life satisfaction to last in the long run . in fact , that would imply better pcs and mcs scores than in the general population . for future studies , examining how favourable outcomes of intensive phases of lifestyle interventions can be maintained , at least partly , over time will be worthwhile . and , in that regard , our finding of continued improvement in the op is of interest . an intervention that can help participants experience less obesity - related problems , despite modest weight loss , may be seen as positive . change of health - related behaviours is challenging and the interaction of time is an aspect that deserves consideration . often , lifestyle interventions are reported to be shorter than the present , for example , four months , six months , or one year [ 4 , 5 ] . the sos study has published data on long - term follow - up , but the length of the included conventional treatment is not standardised or described [ 2 , 8 ] . the look ahead study , though including patients of all weight classes from overweight to severely obese , does provide detailed information about lifestyle intervention for adults with type 2 diabetes and is unique with regard to the large number of participants ( n = 5,145 ) , the randomisation , and the length of the intervention , that is , eight years . clearly , more studies are needed to develop a better understanding of long - term effects of lifestyle interventions on qol outcomes . first , the use of accelerometers to collect data on physical activity increased reliability over self - reporting . second , as recommended , both general and condition - specific qol instruments were used . third , data was collected at four time points and used to reveal associations of patterns of change . since change of health - related behaviour is complex , increase and maintenance of physical activity are challenging , and subjective constructs like qol are not straightforward , these findings on patterns of change contribute uniquely to the body of knowledge about lifestyle interventions for severely obese adults . a limitation of this study was that the number of participants lost to follow - up challenged the statistical power . high attrition is not unusual in research on lifestyle interventions [ 6 , 7 ] but differences between completers and noncompleters vary across studies from none [ 5 , 6 ] to one [ 4 , 7 ] or some . in the present study , the attrition analyses revealed no statistical difference in key variables between the drop - outs and the completers . the secondary analysis confirmed the statistical level of change in the qol measures , counts per minute , and bmi ( data not shown ) . to deal with the challenge of statistical power , all valid data were included in the linear mixed model . regarding the sample size , the study was powered to detect medium sized effect sizes as found in the regression analysis of this study . inclusion of a control group amongst the referred patients was not possible due to their right to treatment . this study examined associations and , therefore , causal relationships could not be inferred . and the study did not control for change of diet , a possible confounder in the associations we examined [ 51 , 52 ] . in addition , we controlled for change in bmi which may be a proxy for diet , because generally diet modifications produce more weight loss than physical activity [ 54 , 55 ] . lastly , the patients were a self - selected , treatment - seeking group although public funding of the intervention gave equal access to all and , therefore , diminished the risk of socioeconomic bias . it has been proposed that lifestyle interventions for obese individuals should focus less on weight loss as the primary outcome and pay more attention to independent benefits of physical activity such as reduction of obesity - related health hazards and improvements of qol . the present study contributes uniquely to the literature on severe obesity , physical activity , and self - reported outcomes and indicates that improved qol may be a valid result of increased physical activity in multicomponent lifestyle interventions . these findings should be further tested in various settings , in larger samples , and with control groups .

neglected tropical diseases constitute a significant public health burden , affecting over one billion people globally , yet this group of diseases is underrepresented in the appropriation of both monetary and intellectual capital for developing improved therapies and public health campaigns . the topic of neglected tropical diseases has been similarly marginalized in the biology classrooms of our nation s high schools and colleges , despite offering an opportunity to teach and learn about a diverse area of microbiology with far - reaching public health , social , and economic implications . discussed herein is an argument for increasing the representation of neglected tropical diseases in microbiology education as a means to generate increased interest in these diseases among the generation of future researchers and policy - makers , and to promote interdisciplinary learning , civic engagement , and critical thinking . it is unknown how changes in physical activity may affect changes in quality of life ( qol ) outcomes during lifestyle interventions for severely obese adults . the purpose of this study was to examine associations in the patterns of change between objectively assessed physical activity as the independent variable and physical , mental , and obesity - specific qol and life satisfaction as the dependent variables during a two - year lifestyle intervention . forty - nine severely obese adults ( 37 women ; 43.6 9.4 years ; body mass index 42.1 6.0 kg / m2 ) participated in the study . assessments were conducted four times using medical outcomes study short - form 36 health survey ( sf-36 ) , obesity - related problems ( op ) scale , a single item on life satisfaction , and accelerometers . the physical component summary ( pcs ) score and the mental component summary ( mcs ) score were used as sf-36 outcomes . associations were determined using linear regression analyses and reported as standardized coefficients ( stand . coeff . ) . change in physical activity was independently associated with change in pcs ( stand . coeff . = 0.35 , p = .033 ) , mcs ( stand . coeff . = 0.51 , p = .001 ) , op ( stand . coeff . = 0.31 , p = .018 ) , and life satisfaction ( stand . coeff . = 0.39 , p = .004 ) after adjustment for gender , age , and change in body mass index .

haemophilus spp . generally colonize the upper respiratory tract and can cause infections such as bronchitis , sinusitis , epiglottitis , pneumonia and meningitis . h. influenzae has been reported as a rare cause of genitourinary tract infection such as urinary tract infection , , , , , pyelonephritis , , prostatitis , epididymitis , salpingitis and endometritis . we report here an immunocompetent japanese man with bacteremic pyelonephritis caused by nontypable h. influenzae associated with a left ureteral calculus . a 44-year - old japanese man with a history of left ureteral renal calculus presented to our hospital with a one - day history of left flank pain , fever and malaise without any respiratory symptoms . he had left flank pain caused by a left ureteral calculus over the past two decades . otherwise , his past history and family history were unremarkable . on physical examination , his blood pressure was 153/91 mm hg , pulse rate 112 beats / min , respiratory rate 20 breaths / min , oxygen saturation on ambient air 98% and body temperature 38.9 c . laboratory tests showed an elevated white blood cell ( wbc ) count 12.2 10/l and creatinine level of 1.25 mg / dl . microscopic examination of the urine sediment showed an elevated wbc and red blood cell ( rbc ) count of 50 and 99 per high power field , respectively . two sets of blood cultures were obtained using bactec plus aerobic / f and anaerobic / f culture bottles ( becton , dickinson and company , sparks , md ) . urine culture was performed using sheep blood agar and drygalski improved medium ( eiken chemical co , ltd , tokyo , japan ) . abdominal ultrasound and computerized tomography ( ct ) scan of the abdomen and pelvis without contrast showed a left ureteral calculus ( 17 10 19 mm ) , left hydronephrosis and a normal - sized prostate gland . after admission , treatment with intravenous ceftriaxone 2 g every 24 h was initiated and the ureteral stent was inserted into his left ureter . after 32 h of incubation , gram - negative bacilli were isolated from an aerobic blood bottle . both blood and urine cultures using sheep blood agar and drygalski improved medium showed no growth . chocolate ii agar ( nissui pharmaceutical co , ltd , tokyo , japan ) for blood and urine based on candle jar method at 37 c were used for culture . the bacteria required both x factor ( hemin ) and v factor ( nicotinamide adenine dinucleotide ) for its growth . , the united states ) automated identification and susceptibility testing showed 99.99% probability of -lactamase - negative h. influenzae biotype 3 . it was also identified as nontypable h. influenzae by influenza bacillus immune serum kit ( denka seiken , co. , ltd , niigata , japan ) . this organism was susceptible to ampicillin ( mic = 0.25 g / ml ) , amoxicillin clavulanic acid ( mic 1 g / ml ) , ampicillin sulbactam ( mic 0.5 g / ml ) , ceftriaxone ( mic 0.12 g / ml ) , meropenem ( mic 0.12 g / ml ) , clarithromycin ( mic = 4 g / ml ) , levofloxacin ( mic 0.12 g / ml ) and rifampicin ( mic 0.5 g / ml ) and resistant to sulfamethoxazole - trimethoprime ( mic = 4 g / ml ) . nasopharyngeal culture that was obtained after the initiation of antimicrobials was negative for haemophilus spp . follow up blood and urine cultures were negative . on hospital day 4 , he became afebrile . he was discharged home on hospital day 9 and treated with oral amoxicillin 1000 mg three times a day for 5 more days . fifteen days after discharge , he underwent extracorporeal shock wave lithotrity for the left ureteral calculus . the non - typable strains generally are not invasive and only the type b strain is preventable by vaccine . h. influenzae has been reported as a rare cause of genitourinary tract infection and fewer case reports of genitourinary tract infections along with bacteremia due to h. influenzae . the reported prevalence of h. influenzae in urine samples was 0.04% in women and 0.22% in men . true prevalence of this organism in the genitourinary tract , may be underestimated , since culture media for this organism are not routinely used for urine specimens . when chocolate agar was used for routine urine cultures showed that biotype 4 predominated in genitourinary isolates , while biotype 1 in respiratory isolates . wallace et al . also reported that nontypable h. influenzae of biotype 4 may have been most common for genitourinary tract infection of women . the results of these studies suggest that h. influenzae biotype 4 might have some mechanism related to the affinity to the genitourinary systems and/or direct invasion to the epithelium . the patients were stratified by gender due to differences in clinical features . in women , 90% ( 18/20 cases ) of the patients were associated with pregnancy and , notably , 75% ( 15/20 ) had bacteremia , . non - bacteremic urinary tract infection was only a case reported by morgan et al . . in men , 25.6% ( 10 out of 39 cases ) of past reported patients had anatomical or functional abnormality of genitourinary tract such as renal calculus , benign prostate hypertrophy , ureteral occlusion or post - operation . there were only 3 cases of bacteremia among the infections in males ( 7.6% ) . therefore the true proportion of the patients with underlying diseases was possibly underestimated . regarding the serotypes of h. influenzae , a total of 66.7% ( 26 out of 39 ) was type b and 28.2% ( 11 out of 39 ) was nontypable ( 2 cases was unknown ) . in 29 out of 39 patients , biologic type of h. influenzae thirteen were type 2 , 9 were type 3 , type 1 or 4 for 3 patients respectively and type 8 was only from one patient . our patient had left renal calculus and serologic / biologic type was nontypable / type 3 . regardless gender and serologic type , h. influenzae were mostly biotype 14 ( 39 out of 42 ) . it is possible that this ability might facilitate colonization or infection in the genitourinary tract . indeed , urease production in the urine will produce an alkaline ph which can predispose to stone formation . we report an immunocompetent patient with a ureteral calculus associated with bacteremic urinary tract infection caused by nontypable h. influenzae ( biotype 3 ) . h. influenzae is difficult to grow on urine culture and its isolation may be underestimated . collaboration between clinicians and microbiology laboratory personnel is essential for correct identification of the organism and appropriate therapy for genitourinary tract infections due to this organism . the long - term survival of patients with hematological malignancies has improved dramatically over the past decades . nowadays , about 40% of patients with acute leukemia or high - grade non - hodgkin lymphoma survive for more than 5 years and about 30% of these patients can be cured . unfortunately , owing to their underlying disease or treatment or both , these patients are at high risk of severe complications , often requiring transfer to the icu . historically , intensivists have been reluctant to admit these patients to the icu because of the almost uniformly fatal prognosis reported in the literature in patients with evolving organ dysfunction requiring mechanical ventilation , vasopressors , or renal replacement therapy alone or in combination . over the past decade , several centers around the world that specialize in the management of these patients have clearly shown that these grim prognostic estimates no longer hold and that the reluctance to admit these patients to the icu , simply because of their underlying malignancy , is no longer justified . an important remaining question , however , is how these patients perform in the long term with regard to survival and quality of life . in a study in the previous issue of critical care , bernal and colleagues focused on the determinants of survival beyond 1 year in a multicenter setting . as could be expected from what we observed at the bedside , functional status ( eastern cooperative oncology group performance status of more than 2 ) , relapsing hematological malignancy , and absence of compliance with the scheduled therapy for the underlying disease after icu discharge were associated with a worse survival . however , what was less expected is that the survival reached nearly 0% after 1.5 years if only one of these factors was present . of note , only 62 patients were included in this study , and depending on whether we focus on a half - empty or half - full glass , the other half of the patients achieved a 5-year post - icu survival of 40% to 50% . in the largest study ever published , including more than 1,000 patients with hematological malignancies admitted over a 16-month period in 17 specialized centers in france and belgium , hospital survival was 60.7% ; up to 80% of these patients had no change in treatment intensity , and 80% were in complete or partial remission 6 months after icu discharge . moreover , recent studies have shown that icu admission does not influence long - term outcome in patients with acute myeloid leukemia who survive the first 30 days after icu discharge : they had similar survival and complete remission rates up to 3 years and 6 years , respectively , after discharge in comparison with acute myeloid leukemia patients for whom icu admission was not necessary . therefore , what the study by bernal and colleagues shows above all is that being technically skilled in advanced life - support therapies is not enough to improve long - term outcome ; as intensivists , we also have to acknowledge better when to use these therapies and when we have to withdraw them during icu stay . more than ever , long - term estimations with regard to survival and quality of life should be taken into account upon referral to the icu . only close collaboration and in - depth communication between hematologists and intensivists upon referral and during icu stay can bridge the two extremes of the overoptimistic oncologists who often overestimate the long - term survival of their patients in daily practice and the overpessimistic intensivists who are reluctant to admit them . such an open and constructive atmosphere , in which physicians assume a leading role , disseminate a clear vision , and let other team members , the patients , and relatives actively and safely participate in the decision - making processes , will not only improve the average long - term outcome of published series focusing on any severe underlying comorbidity but also reduce the burden for individual patients and their relatives at the bedside . health - care workers will also benefit , since real or perceived disproportional care in the icu leads to acute or , much worse , more subtle chronic conflicts within the team , resulting in poor quality of care . the latter is particularly deleterious since it will affect the patient s short- and long - term outcome in general , regardless of whether the admission is justified or not . a good admission policy is necessary in order to safeguard the quality of icu care provided to patients with good long - term expectations on the one hand and to reduce the burden for patients and relatives with poor long - term expectations on the other . this can be achieved only by creating working environments enhancing close collaboration and communication between intensivists and hematologists and where the patient and relatives are closely involved in the decision - making process upon icu referral and during icu stay . it is important to note that this holds not only for patients with hematological malignancies such as in the study by bernal and colleagues but also for patients with any other severe underlying comorbidity that are increasingly referred to the icu . a good admission policy is necessary in order to safeguard the quality of icu care provided to patients with good long - term expectations on the one hand and to reduce the burden for patients and relatives with poor long - term expectations on the other . this can be achieved only by creating working environments enhancing close collaboration and communication between intensivists and hematologists and where the patient and relatives are closely involved in the decision - making process upon icu referral and during icu stay . it is important to note that this holds not only for patients with hematological malignancies such as in the study by bernal and colleagues but also for patients with any other severe underlying comorbidity that are increasingly referred to the icu .

haemophilus species are known to colonize the upper respiratory tract and can cause infections . however haemophilus influenzae has been rarely described as a cause of genitourinary tract infection . we report a 44-year - old nonimmunocompromised japanese man with bacteremic pyelonephritis caused by a nontypable h. influenzae associated with a left ureteral calculus . the organism was isolated from both blood and urine cultures . treatment consisted of 14 days of intravenous ceftriaxone and oral amoxicillin one after than other and insertion of a left ureteral stent . after discharge , he underwent extracorporeal shock wave lithotrity for the left ureteral calculus . he had no recrudescence of the symptoms . h. influenzae should be considered as a genitourinary pathogen among patients with certain risk factors such as anatomical or functional abnormality of genitourinary tract . collaboration between clinicians and microbiology laboratory personnel is essential for correct identification of the organism and appropriate therapy for genitourinary tract infections due to this organism . the spectacular improvement in long - term prognosis of patients with hematological malignancies since the 1980s , coupled with the subsequent improvement over the past decade in short- and mid - term survival in cases of critical illness , resulted in an increasing referral of such patients to the icu . a remaining question , however , is how these patients perform in the long term with regard to survival and quality of life . here we discuss the present multicenter study on survival beyond 1 year in critically ill patients with hematological malignancies . we conclude with suggestions on how we can further improve the long - term outcome of these patients .

chronic hepatitis c ( chc ) is a major health care burden and a leading cause of end - stage liver disease and hepatocellular carcinoma ( hcc ) worldwide . although non - uniform distributions of chc in certain areas complicate the establishment of global and regional epidemiology , the global prevalence of chc has been estimated as 2.35% , affecting approximately 160 million people ( 1 ) . in taiwan , seroprevalences of hepatitis c virus ( hcv ) antibody positivity were estimated as 4.4% to 8.6% ( 2 , 3 ) . upwards of 53% of chc patients are infected with hcv genotype 1 ( g1 ) ( 4 ) . either in most resource - limited areas worldwide or in most of the asian countries , pegylated interferon ( pegifn ) plus ribavirin ( rbv ) combination therapy remains the first - line standard of care ( soc ) for chc . the national health insurance program of taiwan reimburses the cost of soc for chc . however , severe rbv - induced hemolytic anemia severely complicates chc patients during soc by causing suboptimal tolerance , poor compliance and early withdrawal from therapy ( 5 ) . the risks of anemia have been shown to be higher in asian than non - asian patients ( 6 ) . extensive accumulation of rbv in erythrocytes causes membrane oxidative damage , premature hemolysis and subsequent anemia ( 7 ) . the haptoglobin phenotype , pretreatment platelet level ( 8) , impaired renal function ( 9 ) , high dose / body weight ratio , old age and female sex ( 10 ) have been previously demonstrated to be associated with anemia . recent genome - wide association studies have advanced to a strong association between rbv - induced anemia and single - nucleotide polymorphisms ( snps ) in the inosine triphosphate pyrophosphatase ( itpa ) gene on chromosome 20 in chc patients on soc ( 11 ) . identifying patients at high risk of anemia is crucial for improving patient compliance and soc outcomes for chc patients ( 12 ) . novel treatment strategies or algorithms based on a combination of relevant pharmacogenetics and host factors may facilitate patient counseling prior to anemia events ( 13 ) . however , limited studies included predictive modeling of severe anemia ( hemoglobin , hb < 10 g / dl ) based on an index incorporating the strong predictor , itpa snp status ( 14 , 15 ) . therefore , we estimated the effect of itpa snp status on severe anemia and treatment responses to construct a clinically practical predictive index for severe anemia during chc combination therapy . the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki and was approved by the institutional review board of china medical university hospital . informed consent was obtained from each patient included in the study . from september 2005 to september 2013 , consecutive east asian patients with chc g1 were screened and enrolled in a prospective cohort to analyze antiviral treatment responses . we defined chc g1 as positive result for serum anti - hcv antibody ( abbott laboratories , abbott park , il , usa ) for more than six months with detectable serum hcv g1 rna ( cobas amplicor hcv monitor 2.0 ; roche diagnostics , branchburg , nj , usa ) . patients with a history of any of the following conditions were excluded from the cohort ; age < 20 years or > 75 years , treatment discontinued in less than four months after starting soc with no severe anemia event , hepatitis b virus coinfection , human immunodeficiency virus coinfection , hcc , alcoholic liver disease , primary biliary cirrhosis , primary sclerosing cholangitis , wilson s disease , autoimmune hepatitis , hemochromatosis , previous ifn - based therapy , baseline hb < 13 g / dl in men , hb < 12 g / dl in women , nucleoside- or nucleotide - analog therapies other than rbv , decompensated cirrhosis and end - stage renal disease . eligible patients were randomly assigned to the development and validation cohorts at a ratio of 4:1 . patients received pegifn -2a ( pegasys , hoffmann - la roche , basel , switzerland ) at a dosage of 180 g / week or pegifn -2b ( peg - intron , schering - plough , kenilworth , nj , usa ) at a dosage of 1.5 g / kg / week subcutaneously . oral rbv was prescribed for all patients at a daily dose of 1000 mg ( body weight < 75 kg ) or 1200 mg ( body weight 75 kg ) for 24 weeks or 48 weeks based on the baseline hcv rna ( < versus 6 log10 copies / ml ) and virological response at week 4 ( 16 , 17 ) . baseline and mean doses of rbv ( mg / kg / day ) on treatment were calculated for each patient . erythropoietin ( epo ) was considered if hb fell below 10 g / dl during the treatment . blood biochemistry ( beckman coulter , ca , usa ) and complete blood count analyses ( sysmex hst - series , kanogawa , japan ) were performed in the central laboratory at the medical center . hcv rna load was quantified at baseline and monitored at weeks 4 , 12 , 24 and 48 . sustained virological response ( svr ) was defined as undetectable hcv rna at or before the end of treatment and at 24 weeks after the end of treatment . monthly distributions of patients with severe anemia ( hemoglobin < 10 g / dl ) on treatment were compared between c / c versus a / a or c / a genotypes . participant genomic dna was extracted from peripheral blood mononuclear cells using a qiagen dna blood mini kit ( qiagen , valencia , ca , usa ) . genotyping for one functional missense itpa variant in exon 2 at rs1127354 on chromosome 20 ( 18 , 19 ) was performed using 20 l of faststart universal probe master mix ( roche diagnostics , branchburg , nj , usa ) containing 500 nmol rs1127354 forward primer ( 5-tcttggaacaggtcgttcagattcta-3 ) , 500 nmol rs1127354 reverse primer ( 5-aggaagacagagaaatccaaccatc-3 ) , 250 nmol c allele probe ( fam - agtttccatgcactttggtgg - bbq ) and 250 nmol a allele probe ( yak - agtttacatgcactttggtggc - bbq ) . the reaction mixture was denatured at 95c for 10 minutes before thermal cycling at 95c for 15 seconds and 60c for 1 minute for 40 cycles . genotypes were analyzed using steponetm software version 2.0 ( life technologies , carlsbad , ca , usa ) . therefore , patients were not genotyped for itpa snp rs7270101 ( 18 ) . the cohort was also genotyped for interleukin 28b ( il28b ) polymorphisms at rs8099917 and rs12979860 , as previously described ( 20 ) . both kolmogorov - smirnov and shapiro - wilk tests showed that ( p < 0.05 ) none of the continuous variables in this study had a normal distribution . therefore , continuous variables were expressed as median ( interquartile range , iqr ) and estimated using mann - whitney u test . categorical variables were estimated using the chi - square test or fisher s exact test . univariate logistic regression identified the variables ( p < 0.25 ) associated with severe anemia for subsequent multiple regressions . by expressing the likelihood of severe anemia as a probability ranging from 0 to 1 , an index for identifying severe anemia was constructed by incorporating significant independent associated factors and the beta coefficients acquired through the final multiple logistic regression for severe anemia ( 14 ) . the diagnostic performance of the predictive index was evaluated by the area under the receiver operating - characteristic curves ( aucs ) . the odds ratios ( ors ) of significant associations were determined based on a 95% confidence interval ( ci ) . the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki and was approved by the institutional review board of china medical university hospital . from september 2005 to september 2013 , consecutive east asian patients with chc g1 were screened and enrolled in a prospective cohort to analyze antiviral treatment responses . we defined chc g1 as positive result for serum anti - hcv antibody ( abbott laboratories , abbott park , il , usa ) for more than six months with detectable serum hcv g1 rna ( cobas amplicor hcv monitor 2.0 ; roche diagnostics , branchburg , nj , usa ) . patients with a history of any of the following conditions were excluded from the cohort ; age < 20 years or > 75 years , treatment discontinued in less than four months after starting soc with no severe anemia event , hepatitis b virus coinfection , human immunodeficiency virus coinfection , hcc , alcoholic liver disease , primary biliary cirrhosis , primary sclerosing cholangitis , wilson s disease , autoimmune hepatitis , hemochromatosis , previous ifn - based therapy , baseline hb < 13 g / dl in men , hb < 12 g / dl in women , nucleoside- or nucleotide - analog therapies other than rbv , decompensated cirrhosis and end - stage renal disease . eligible patients were randomly assigned to the development and validation cohorts at a ratio of 4:1 . patients received pegifn -2a ( pegasys , hoffmann - la roche , basel , switzerland ) at a dosage of 180 g / week or pegifn -2b ( peg - intron , schering - plough , kenilworth , nj , usa ) at a dosage of 1.5 g / kg / week subcutaneously . oral rbv was prescribed for all patients at a daily dose of 1000 mg ( body weight < 75 kg ) or 1200 mg ( body weight 75 kg ) for 24 weeks or 48 weeks based on the baseline hcv rna ( < versus 6 log10 copies / ml ) and virological response at week 4 ( 16 , 17 ) . baseline and mean doses of rbv ( mg / kg / day ) on treatment were calculated for each patient . erythropoietin ( epo ) was considered if hb fell below 10 g / dl during the treatment . blood biochemistry ( beckman coulter , ca , usa ) and complete blood count analyses ( sysmex hst - series , kanogawa , japan ) were performed in the central laboratory at the medical center . hcv rna load was quantified at baseline and monitored at weeks 4 , 12 , 24 and 48 . sustained virological response ( svr ) was defined as undetectable hcv rna at or before the end of treatment and at 24 weeks after the end of treatment . monthly distributions of patients with severe anemia ( hemoglobin < 10 g / dl ) on treatment were compared between c / c versus a / a or c / a genotypes . participant genomic dna was extracted from peripheral blood mononuclear cells using a qiagen dna blood mini kit ( qiagen , valencia , ca , usa ) . genotyping for one functional missense itpa variant in exon 2 at rs1127354 on chromosome 20 ( 18 , 19 ) was performed using 20 l of faststart universal probe master mix ( roche diagnostics , branchburg , nj , usa ) containing 500 nmol rs1127354 forward primer ( 5-tcttggaacaggtcgttcagattcta-3 ) , 500 nmol rs1127354 reverse primer ( 5-aggaagacagagaaatccaaccatc-3 ) , 250 nmol c allele probe ( fam - agtttccatgcactttggtgg - bbq ) and 250 nmol a allele probe ( yak - agtttacatgcactttggtggc - bbq ) . the reaction mixture was denatured at 95c for 10 minutes before thermal cycling at 95c for 15 seconds and 60c for 1 minute for 40 cycles . genotypes were analyzed using steponetm software version 2.0 ( life technologies , carlsbad , ca , usa ) . the cohort was also genotyped for interleukin 28b ( il28b ) polymorphisms at rs8099917 and rs12979860 , as previously described ( 20 ) . both kolmogorov - smirnov and shapiro - wilk tests showed that ( p < 0.05 ) none of the continuous variables in this study had a normal distribution . therefore , continuous variables were expressed as median ( interquartile range , iqr ) and estimated using mann - whitney u test . categorical variables were estimated using the chi - square test or fisher s exact test . univariate logistic regression identified the variables ( p < 0.25 ) associated with severe anemia for subsequent multiple regressions . by expressing the likelihood of severe anemia as a probability ranging from 0 to 1 , an index for identifying severe anemia was constructed by incorporating significant independent associated factors and the beta coefficients acquired through the final multiple logistic regression for severe anemia ( 14 ) . the diagnostic performance of the predictive index was evaluated by the area under the receiver operating - characteristic curves ( aucs ) . the odds ratios ( ors ) of significant associations were determined based on a 95% confidence interval ( ci ) . excluded patients included those with baseline hepatitis b virus coinfection ( n = 9 ) , hcc ( n = 5 ) , a history of ifn - based therapy ( n = 11 ) , men with baseline hb < 13 g / dl or women < 12 g / dl ( n = 8) and end - stage renal disease ( n = 2 ) . therefore , the chc g1 cohort consisted of 418 eligible participants who intended to receive combination therapy . nine patients were also excluded , because the treatment was discontinued within four months from the treatment baseline for reasons other than severe anemia . the cohort was randomized into a development cohort ( n = 324 ) and a validation cohort ( n = 81 ) . among them , 367 ( 90.6% ) of 405 patients had liver histology data . a total of 148 ( 36.5% ) patients received combination therapy for 24 weeks and 257 ( 63.5% ) patients for 48 weeks . patients per protocol ( n = 357 ) entered the analysis for svr ( 41 patients discontinued before the intended eot and seven patients were lost to follow - up for svr visit ) . the median patient age was 53.0 ( 13.0 ) years and 49.6% of patients were men . the c / c genotype was present in 66.4% of patients and 33.6% had the a / a or the c / a allelic variants ( a / a or c / a as a combo variable ) at rs1127354 . the development cohort ( n = 324 ) and validation cohort ( n = 81 ) did not differ significantly in a univariate analysis . monthly distributions of severe anemia events are demonstrated in figure 1 . among patients who received 24-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month on treatment than those with a / a or c / a genotype ( 37.1% versus 0.0% , p = 0.0135 ) . likewise , among patients who received 48-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month on treatment than those with a / a or c / a genotype ( 22.9% versus 2.9% , p = 0.0079 ) . in total , 166 patients ( 166/324 , 51.2% ) in the development cohort developed severe anemia on treatment . multiple logistic regressions identified age ( 50 y : or = 9.7 , 95% ci = 5.0 - 18.6 ) , itpa rs1127354 ( c / c : or = 3.3 , 95% ci = 1.8 - 5.8 ) and baseline hemoglobin ( < 14.0 g / dl : or 6.4 , 95% ci = 3.3 - 12.1 ; 14.0 - 14.9 : or = 2.4 , 95% ci = 1.2 - 4.6 ) as predictors of severe anemia on treatment ( tables 1 and 2 ) . there was no significant dose effect between itpa snp status ( c / a versus a / a , p = 0.5387 ) and severe anemia . therefore , the predictive index derived from the development cohort incorporating age ( y ) , itpa snp status and baseline hb ( g / dl ) was expressed as 1/(1 + exp ( - x ) ) , where x = - 3.2835 + 2.2709 a + 1.1822 i + 1.8848 h1 + 0.8684 h2 ( a = 1 if age 50 ; a = 0 if age < 50 ; i = 1 if itpa snp = c / c ; i = 0 if itpa snp = a / a or c / a ; h1 = 1 if hb < 14.0 ; h1 = 0 if hb 14.0 ; h2 = 1 if hb = 14.0 - 14.9 ; h2 = 0 if hb < 14.0 or 15.0 ) . to dichotomize the anemia status ( with versus without severe anemia ) in the development , the cutoff value of 0.5 was optimal when maximizing the values of the predictive index yielded a diagnostic accuracy ( auc ) of 0.830 ( 95% ci = 0.783 - 0.871 ) in the development ( n = 324 ) and 0.902 ( 0.826 - 0.925 ) in the validation ( n = 81 ) cohorts to dichotomize the anemia status , respectively ( figure 2 ) . in total , 135 ( 135/357 per protocol , 37.8% ) patients achieved rvr . seventy - three of them ( 73/135 , 54.1% ) had a median baseline hcv rna of 5.29 ( 0.76 ) log10 copies / ml and received 24-week treatment . the remaining 62 patients ( 62/135 , 45.9% ) received 48-week treatment . liver pathology was acquired in 69 of 73 ( 94.5% ) patients with rvr and 24-week treatment . among them , metavir fibrosis stages 0 - 2 were noted in 54 ( 54/69 , 78.3% ) , and 3 - 4 in 15 ( 15/69 , 21.7% ) . in total , 255 ( 255/357 per protocol , 71.4% ) patients achieved svr . the rvr ( or = 8.2 ; 95% ci = 3.519.1 ; p < .0001 ) , t / t il28b genotype at rs8099917 ( or = 3.0 ; 95% ci = 1.2 - 7.4 ; p = 0.0156 ) , the metavir fibrosis stages 0 - 2 ( or = 1.9 ; 95% ci = 1.0 - 3.5 ; p = 0.0450 ) , baseline hcv rna ( or = 0.4 ; 95% ci = 0.2 - 0.7 ; p = 0.0008 ) and 48-week treatment duration ( or = 1.1 ; 95% ci = 1.0 - 1.1 ; p = 0.0003 ) were independently associated with svr . anemia events and epo treatment were not independently associated with svr . in patients with either itpa c / c or a / a or c / a genotypes , the mean rbv dose exposures were not significantly different between patients with svr versus non - svr . the il28b snp ( rs12979860 ) was not included in the final multiple regression because of collinearity with the other il28b snp ( rs8099917 ) ( table 3 ) . in addition , through univariate analyses , the itpa snp status was significantly associated with rbv dose reduction in the development cohort ( n = 324 ) ( mann - whitney u test , p = 0.0035 ) . patients ( n = 103/324 , 31.8% ) with a / a or c / a genotype received 100% ( 8% ) intended rbv doses . in contrast , patients ( n = 221/324 , 68.2% ) with c / c genotype received 97% ( 1.4% ) intended rbv doses . however , itpa snp status was not significantly associated with epo use ( chi - square test , p = 0.1617 ) . forty - five of 103 patients ( 45/103 , 43.7% ) with a / a or c / a genotype received epo treatment . in contrast , 115 of 221 patients ( 115/221 , 52.0% ) with c / c genotype received epo treatment . a ) among patients who received 24-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month than those with a / a or c / a genotype ( p = 0.0135 ) . b ) ; also , among patients who received 48-week treatment ( p = 0.0079 ) . data are presented for hemoglobin < 10 g / dl and development cohort ( n = 324 ) . excluded patients included those with baseline hepatitis b virus coinfection ( n = 9 ) , hcc ( n = 5 ) , a history of ifn - based therapy ( n = 11 ) , men with baseline hb < 13 g / dl or women < 12 g / dl ( n = 8) and end - stage renal disease ( n = 2 ) . therefore , the chc g1 cohort consisted of 418 eligible participants who intended to receive combination therapy . nine patients were also excluded , because the treatment was discontinued within four months from the treatment baseline for reasons other than severe anemia . the cohort was randomized into a development cohort ( n = 324 ) and a validation cohort ( n = 81 ) . among them , 367 ( 90.6% ) of 405 patients had liver histology data . a total of 148 ( 36.5% ) patients received combination therapy for 24 weeks and 257 ( 63.5% ) patients for 48 weeks . patients per protocol ( n = 357 ) entered the analysis for svr ( 41 patients discontinued before the intended eot and seven patients were lost to follow - up for svr visit ) . the median patient age was 53.0 ( 13.0 ) years and 49.6% of patients were men . the c / c genotype was present in 66.4% of patients and 33.6% had the a / a or the c / a allelic variants ( a / a or c / a as a combo variable ) at rs1127354 . the development cohort ( n = 324 ) and validation cohort ( n = 81 ) did not differ significantly in a univariate analysis . monthly distributions of severe anemia events are demonstrated in figure 1 . among patients who received 24-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month on treatment than those with a / a or c / a genotype ( 37.1% versus 0.0% , p = 0.0135 ) . likewise , among patients who received 48-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month on treatment than those with a / a or c / a genotype ( 22.9% versus 2.9% , p = 0.0079 ) . in total , 166 patients ( 166/324 , 51.2% ) in the development cohort developed severe anemia on treatment . multiple logistic regressions identified age ( 50 y : or = 9.7 , 95% ci = 5.0 - 18.6 ) , itpa rs1127354 ( c / c : or = 3.3 , 95% ci = 1.8 - 5.8 ) and baseline hemoglobin ( < 14.0 g / dl : or 6.4 , 95% ci = 3.3 - 12.1 ; 14.0 - 14.9 : or = 2.4 , 95% ci = 1.2 - 4.6 ) as predictors of severe anemia on treatment ( tables 1 and 2 ) . there was no significant dose effect between itpa snp status ( c / a versus a / a , p = 0.5387 ) and severe anemia . therefore , the predictive index derived from the development cohort incorporating age ( y ) , itpa snp status and baseline hb ( g / dl ) was expressed as 1/(1 + exp ( - x ) ) , where x = - 3.2835 + 2.2709 a + 1.1822 i + 1.8848 h1 + 0.8684 h2 ( a = 1 if age 50 ; a = 0 if age < 50 ; i = 1 if itpa snp = c / c ; i = 0 if itpa snp = a / a or c / a ; h1 = 1 if hb < 14.0 ; h1 = 0 if hb 14.0 ; h2 = 1 if hb = 14.0 - 14.9 ; h2 = 0 if hb < 14.0 or 15.0 ) . to dichotomize the anemia status ( with versus without severe anemia ) in the development , the cutoff value of 0.5 was optimal when maximizing the values of the predictive index yielded a diagnostic accuracy ( auc ) of 0.830 ( 95% ci = 0.783 - 0.871 ) in the development ( n = 324 ) and 0.902 ( 0.826 - 0.925 ) in the validation ( n = 81 ) cohorts to dichotomize the anemia status , respectively ( figure 2 ) . seventy - three of them ( 73/135 , 54.1% ) had a median baseline hcv rna of 5.29 ( 0.76 ) log10 copies / ml and received 24-week treatment . liver pathology was acquired in 69 of 73 ( 94.5% ) patients with rvr and 24-week treatment . among them , metavir fibrosis stages 0 - 2 were noted in 54 ( 54/69 , 78.3% ) , and 3 - 4 in 15 ( 15/69 , 21.7% ) . in total , 255 ( 255/357 per protocol , 71.4% ) patients achieved svr . the rvr ( or = 8.2 ; 95% ci = 3.519.1 ; p < .0001 ) , t / t il28b genotype at rs8099917 ( or = 3.0 ; 95% ci = 1.2 - 7.4 ; p = 0.0156 ) , the metavir fibrosis stages 0 - 2 ( or = 1.9 ; 95% ci = 1.0 - 3.5 ; p = 0.0450 ) , baseline hcv rna ( or = 0.4 ; 95% ci = 0.2 - 0.7 ; p = 0.0008 ) and 48-week treatment duration ( or = 1.1 ; 95% ci = 1.0 - 1.1 ; p = 0.0003 ) were independently associated with svr . anemia events and epo treatment were not independently associated with svr . in patients with either itpa c / c or a / a or c / a genotypes , the mean rbv dose exposures were not significantly different between patients with svr versus non - svr . the il28b snp ( rs12979860 ) was not included in the final multiple regression because of collinearity with the other il28b snp ( rs8099917 ) ( table 3 ) . in addition , through univariate analyses , the itpa snp status was significantly associated with rbv dose reduction in the development cohort ( n = 324 ) ( mann - whitney u test , p = 0.0035 ) . patients ( n = 103/324 , 31.8% ) with a / a or c / a genotype received 100% ( 8% ) intended rbv doses . in contrast , patients ( n = 221/324 , 68.2% ) with c / c genotype received 97% ( 1.4% ) intended rbv doses . however , itpa snp status was not significantly associated with epo use ( chi - square test , p = 0.1617 ) . forty - five of 103 patients ( 45/103 , 43.7% ) with a / a or c / a genotype received epo treatment . in contrast , 115 of 221 patients ( 115/221 , 52.0% ) with c / c genotype received epo treatment . a ) among patients who received 24-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month than those with b ) ; also , among patients who received 48-week treatment ( p = 0.0079 ) . data are presented for hemoglobin < 10 g / dl and development cohort ( n = 324 ) . this incidence was higher than that reported ( 39.3% ) in a previous study of 466 east asian patients diagnosed with chc ( 5 ) . the higher anemia frequency in our study can be attributed to the higher rbv dosage in patients infected with hcv g1 . consistent with hb kinetics reported in previous studies ( 5 , 12 , 19 , 22 - 25 ) , the anemia events in our study accumulated during the first few months of treatment and remained relatively stable thereafter ( figure 1 ) . the accumulated itp was substituted for gtp during the biosynthesis of atp and protected against rbv - induced hemolytic anemia . . methods of gene expression analysis such as mrna transcripts or western blotting were not used in our study . however , the results of snp genotyping of itpa that we performed correlated closely with phenotypes or predicted itpa activities of these allelic variants ( 26 - 28 ) . although rbv pharmacokinetics in serum or in erythrocytes or predicted itpa activities were not applicable in our current study ( 29 ) , this index may serve as a clinically practical one based on dichotomized parameters available in clinical settings . future studies can develop algorithms or scoring systems based on age , sex , body weight , renal function , hb and itpa snp status for modifying rbv dosage to achieve an optimal steady - state concentration range or for administering epo prior to the development of anemia , rather than reducing the rbv dose following an anemia event ( 30 , 31 ) . recent studies indicated that itpa allelic variants a / a or c / a at rs1127354 are less likely associated with rbv dose reduction ( 19 , 22 , 23 , 32 ) . future studies can also use a time - to - event analysis to identify significant predictors for rbv dose reduction . the effects of older age and female sex on severe anemia ( < 11 g / dl ) during the first four weeks of treatment were also reported in a study of 61 hcv g1-infected japanese patients , in which 49 patients exhibited rbv - sensitive itpa c / c genotype at rs1127354 and 12 patients exhibited rbv - resistant a / a or c / a variants ( 19 ) . therefore , older female patients require stringent monitoring of anemia during combination therapy . however , sex was not included in the predictive index through multiple logistic regressions in the current study because of collinearity with baseline hb . therefore , it was not applicable to adopt creatinine level in multiple regression modeling for predictive index of severe anemia . in the study by tsubota et al . , estimated glomerular filtration rate was found to be one of the significant associated factors to construct a predictive model for rbv - induced anemia ( 14 ) . our future studies would recruit participants in larger sample sizes receiving more uniform treatment durations to allow time - dependent analysis of anemia events for constructing a predictive index through cox regression analysis . based on our results , we suggest that subjects with a predictive index greater than 0.5 might receive epo treatment early after the start of pegifn and rbv combination therapy to facilitate quality of life , prevent rbv dose reduction and avoid compromised treatment responses . however , anticipated benefits associated with early implementation of epo treatment need to be confirmed by further studies . both rvr and t / t il28b genotype at rs8099917 remained significant predictors for svr in our chc g1 cohort ( 33 ) . also consistent with recent studies ( 18 , 19 , 22 - 24 , 34 ) , itpa snp status was a non - significant predictor of svr in our study . however , the reported itpa - svr correlations varied ( 35 ) . in a recent report by rembeck et al . ( 27 ) , itpa allelic variants encoding reduced itpa activity was demonstrated to correlate with svr and non - relapse , unrelated to rbv adherence or protection against anemia , although the molecular mechanisms require further elucidation . although previous reports yielded conflicting results regarding the significance of anemia - svr correlation ( 5 , 12 , 36 , 37 ) , most recent studies on itpa snps have not estimated anemia - svr correlation ( 18 , 19 , 22 - 25 , 32 , 38 ) . in our study , svr was not associated with severe anemia or hb decline ( 4 g / dl ) on treatment ( table 3 ) . recent anemia - svr correlations have also been reported based on the divergent results of non - genetic ( 5 , 23 , 36 ) and molecular studies ( 11 , 14 ) . molecular studies indicated that anemia might not only be a surrogate for rbv exposure , because anemia - svr association was attenuated when adjusting for rbv levels ( 39 ) , but also represent the first molecular evidence connecting anemia - svr association to novel genes and pathways through statistical analyses on gene expressions and mrna signatures ( 11 ) . in conclusion , itpa snp status is a significant predictor of rbv - induced hemolytic anemia in patients diagnosed with chc g1 and receiving pegifn and rbv combination therapy . the itpa snp - based index is an accurate and practical predictive solution for severe anemia in clinical practice . information on itpa snp status of patients may assist in maximizing the tolerability of soc for patients with chc . endometrial carcinoma is the most common cancer of the female genital tract in the western world . endometrial carcinomas are generally thought to have a favorable prognosis due to early detection , and the majority of tumors are detected in early stages . however , in fact this is not fully true , and there are important subgroups within this diagnosis with poor prognosis and outcome of treatment . therefore , the first step to improve the situation has been to find predictive and prognostic factors , then to define clinically relevant risk groups , and finally to design clinical trials and treatment options for these risk groups . unfortunately , no consensus exists on which predictive or prognostic factors that should be used and how to combine them in the definition of suitable - risk groups . as a result of this , the randomized phase iii trials presented during the last decades are difficult to compare since these definitions have varied , more or less , in most of them . another problem has been the small size and low power of most studies in the literature dealing with prognostic and predictive factors . despite more or less sophisticated statistical methods with multivariate technique , the results are not reliable enough for definitive conclusions from such small series analyzing multiple variables . a few exceptions do exist but then with data from large registry studies , but then with other problems of selection and bias built in . six prospective randomized studies have been presented since 1980 to elucidate the value of external beam pelvic radiotherapy after surgery in early - stage endometrial carcinoma ( aalders , portec-1 , gog#99 , astec / en.5 , portec-2 , and sorbe ) [ 17 ] . the treated populations varied in all these studies from no risk groups defined ( aalders ) to a mixture of low - risk ( portec-1 , gog#99 ) [ 2 , 3 ] , medium - risk ( portec-1 , gog#99 , astec / en.5 , and portec-2 ) [ 25 ] , or high - risk cases ( astec / en.5 ) . type of primary surgery and staging also varied from no staging at all ( aalders , portec-1 , astec / en.5 , and portec-2 ) [ 1 , 2 , 4 , 5 ] to staging with lymph node sampling or complete lymphadenectomy ( gog#99 , astec / en.5 ) [ 3 , 4 ] . subgroup analyses performed within the frame of these studies have suffered from low power , and no level one data are presented for well - defined medium - risk or high - risk groups . three prospective randomized trials of low - risk , medium - risk , and high - risk cancers have been performed in sweden and some other european countries . vaginal brachytherapy , external beam pelvic radiation , and adjuvant chemotherapy were addressed in these studies . study a large , comprehensive , and consecutive series of more than 4,500 endometrial carcinomas in figo stages i iv were analyzed with regard to predictive and prognostic factors and definition of the risk groups used in the above mentioned three prospective randomized studies . special emphasis will be made on the prognostic value of dna ploidy and the importance of this factor in the risk group definitions . one swedish cancer center ( rebro ) for gynecological oncology recruited patients with all stages ( figo i iv ) of endometrial carcinomas in an observation study . the period of recruitment was from january 1975 to december 2009 . in all , 4,543 patients were included . postoperative external pelvic irradiation and/or vaginal brachytherapy were administered to the majority of the patients . the definition of high - risk carcinomas was as follows : ( 1 ) figo stage i , ( 2 ) nonendometrioid histological type , ( 3 ) presence of two of the following risk factors : figo grade 3 ( poorly differentiated ) , deep ( 50% ) myometrial infiltration , dna aneuploidy ( fcm ) , ( 4 ) nuclear grade 3 , ( 5 ) pathologically negative lymph nodes , and ( 6 ) negative abdominal cytology . points 5 - 6 were optional in this study , and data are not available for all cases . the definition of medium - risk carcinomas was as follows : ( 1 ) figo stage i , ( 2 ) endometrioid histological type , ( 3 ) presence of one of the following risk factors : figo grade 3 ( poorly differentiated ) , deep ( 50% ) myometrial infiltration , dna aneuploidy ( fcm ) , ( 4 ) nuclear grade 1 - 2 , ( 5 ) pathologically negative lymph nodes , and ( 6 ) negative abdominal cytology . points 5 - 6 were optional in this study , and data are not available for all cases . lymph vascular space invasion ( lvsi ) was not regularly included in the pathology reports at the participating centers and was not included in the definition of the medium - risk group . the definition of low - risk carcinomas was as follows : ( 1 ) figo stage i , ( 2 ) endometrioid histological type , ( 3 ) presence of none of the following risk factors : figo grade 3 ( poorly differentiated ) , deep ( 50% ) myometrial infiltration , dna aneuploidy ( fcm ) , or ( 4 ) nuclear grade 3 . the primary surgery was total abdominal hysterectomy , bilateral salpingo - oophorectomy , appendectomy , node sampling of enlarged lymph nodes , and peritoneal washing with cytology . lymphadenectomy ( pelvic paraaortic ) was not performed as a routine at the centers referring patients to the regional clinic . the surgery was performed at five departments of gynecology and obstetrics , but all patients were then referred to a gynecologic oncology department for postoperative evaluation and treatment . the median follow - up period at the time of analysis was 115 months ( range 1362 months ) for patients alive . during all visits , symptoms and signs related to the therapy were recorded , but in this study treatment - related side effects are not presented . for the brachytherapy treatments , plastic vaginal cylinders with a diameter of 20 mm , 25 mm , or 30 mm were used as standard . the diameter of the cylinder was individually chosen to ensure good contact between the surface of the applicator and the vaginal mucosa . the length of the vagina was measured from the vault to the level of introitus . the dose per fraction was specified at a depth of 5 mm from the surface of the vaginal cylinder with the hdr technique . library dose plans that covered different vaginal lengths in steps of 10 mm and the different diameters of the cylinders were used . the dose calculations were made on the nucletron planning system ( nps v. 10 ) and the plato brachytherapy planning system ( bps v. 14 ) at centers using this equipment . thus , the total doses delivered were 15.018.0 gy . recalculated to 2-gy - equivalent doses ( eqd2 ) , the total doses were 15.619.5 gy at a depth of 5 mm ( / = 10.0 ) . external beam therapy was given to patients with high - risk tumors and to many with medium - risk tumors . the target volume was the previous site of the uterus and adnexa , the parametria , the proximal two - thirds of the vagina , and the lymphatic drainage regions along the iliac vessels up to the promontory . the superior field border was set at the l5-s1 disk . the total dose to be delivered to this volume was 46 gy ( median dose 46.0 gy , range 650 gy ) and daily fractions of 1.82.0 gy ( table 3 ) . all data were collected in a computerized database at the regional oncology center , rebro , sweden . in the statistical analyses , survival curves were generated using the kaplan - meier technique , and differences were tested with the log - rank test . the pearson chi - square test was used for comparison of proportions and the independent t - test for comparing means of two groups . multivariate analysis of prognostic factors was performed using the cox proportional hazards model and logistic regression analysis . best subset analysis was performed with multivariate technique to find the most important prognostic factors and to find the most powerful combination of these factors . all p values were based on two - sided tests , with p < 0.05 considered statistically significant . the statistica software package ( statsoft , inc . , tulsa , ok , usa , version 10 , 2010 ) was used for the statistical analyses . the overall recurrence rate of the complete series was 519 out of 4,543 cases or 11.4% . the regional pelvic ( excluding vaginal recurrence ) recurrence rate was 2.3% ( 103 cases ) , and the locoregional ( vaginal or pelvic , or both ) recurrence rate was 4.2% ( 190 cases ) . distant recurrences ( outside the pelvic area ) were noted in 329 cases ( 7.2% ) , and the 5-year actuarial relapse rate was 6.6% . the median time to relapse in in the complete series , 370 out of 519 recurrences ( 71% ) occurred within 3 years and 445 recurrences ( 86% ) within 5 years . the median age of all patients was 67.0 years ( 2399 years ) , for those with recurrences was 68.4 years , and those without recurrences was 66.4 years . eight of these factors ( age , figo stage , histology , figo grade , nuclear grade , dna ploidy , myometrial infiltration , and p53 expression ) were analyzed in this study with regard to the risk of tumor recurrence , both total rate and locoregional and distant recurrences . in a multivariate logistic regression analysis , three of these factors ( figo grade , depth of myometrial infiltration , and dna ploidy ) were independent and statistically significant with regard to overall recurrence rate and distant recurrences ( table 5 ) . the fourth most important predictive factor was the nuclear grade ( best subset analysis ) . for locoregional recurrences no significant results were noted for these risk factors . in a model building analysis with best subset technique , the figo grade , depth of myometrial infiltration , and dna ploidy gave the best predictive information with regard to the risk of tumor recurrences . addition of further factors ( age , histology , nuclear grade ) only marginally increased the predictive value of the model . depth of myometrial infiltration was the second most important , and dna ploidy ( aneuploidy ) the third factor . in this series 23.7% of the tumors with evaluable dna status ( n = 1,613 ) were nondiploid ( aneuploid ) . at the last followup ( march 2010 ) , the number of patients alive was 2,764 ( 61% ) , dead of disease 819 ( 18% ) , and dead of intercurrent disease 960 ( 21% ) . the five - year actuarial overall survival rate was 73% and the cancer - specific survival rate was 83% . the salvage rate was 44% ( 38/87 ) after isolated vaginal recurrences , 20% ( 21/103 ) after pelvic recurrences , and 6% ( 19/329 ) after distant recurrences . eight prognostic factors were analyzed with cox proportional multivariate regression analyses and with overall and cancer - specific survival rate as the dependent variable . tumor stage ( stages iii - iv versus i - ii ) was the single most important factor with a risk ratio of 4.2 ( 95% ci 3.55.0 ) for advanced tumor stage . tumor grade ( grade 3 versus 1 - 2 ) was the second most important prognostic factor with risk ratio 2.5 ( 95% ci 2.13.0 ) . depth of myometrial infiltration had the lowest risk ratio 1.3 ( 95% ci 1.11.6 ) among the seven significant risk factors . the nuclear grade of the tumor was significant and independent of the figo grade in multivariate analysis . dna ploidy ( aneuploid versus diploid ) was also an important and significant prognostic factor with a risk ratio of 1.6 ( 95% ci 1.32.0 ) with regard to cancer - specific survival rate . the risk group definitions presented under material and methods were used in the complete series and for all stages together and for stage i alone . in the complete series , 54% of the cases fulfilled low - risk criteria , 23% medium - risk criteria , and 22% high - risk criteria . in stage i , the corresponding figures were 57% , 25% , and 17% , respectively . the discriminating power ( chi - square = 471.8 ; p < 0.000001 ) with regard to cancer - specific survival rate was very high both for the complete series and for stage i tumors alone . the 5-year survival rate in the high - risk group was only 50% in the complete series , and this group was very distinctly separated from the low - risk and medium - risk groups ( figure 1 ) . on the other hand , the difference in survival between the last two groups was only 10% at 5 years . if instead only two risk groups are used , which are proposed for preoperative risk group definitions , a 30% difference was noted in cancer - specific survival at 5 years , which was also highly statistically significant ( z = 22.948 ; p < 0.000001 ) . in figo stage i , the difference between the two groups was 20% ( 75% versus 95% ) ( z = 12.980 ; p < 0.000001 ) ( figure 2 ) . in the preoperative definitions , only three prognostic factors were used : histology ( nonendometrioid versus endometrioid ) , figo grade ( grade 3 versus 1 - 2 ) , and dna ploidy ( nondiploid versus diploid ) . it was not necessary to use myometrial infiltration , which is not a reliable prognostic factor assessed preoperatively . the optimal treatment of endometrial carcinoma patients have been vividly discussed and also studied in a number of randomized trials during the last decades [ 19 ] . before that , no consensus existed with regard to type of therapy , but the situation has changed , and our evidence - based knowledge in this field has improved substantially . however , still different conclusions are drawn from the available study data , and the optimal treatment of the various risk groups is continuously debated in various countries and in different centers . various definitions of the risk groups have confused the results , and conclusions drawn from the studies [ 25 , 79 ] . the portec-1 study included both low - risk ( grade 2 , superficial infiltration ) and medium - risk cases , and the astec / en.5 study included both medium - risk and high - risk cases . most authors agree that low - risk cases can be left with surgery alone [ 7 , 10 , 11 ] . still , vaginal brachytherapy is effective and will reduce the rate of vaginal recurrences in all risk groups , but from different levels . from a cost - effectiveness perspective , it seems reasonable to exclude the low - risk tumors from this type of adjuvant therapy . treatment of isolated vaginal relapse after surgery alone is effective in 89% ( complete remission ) with 65% survival rate . in the present study , the salvage rate was only 44% after vaginal recurrences . for medium - risk cases , a number of studies have focused on this risk group , but still with various definitions of this risk group ( medium - risk , low - medium risk , and high - medium risk ) [ 19 ] . a mixture of both low - risk and medium - risk cases has been studied as well as a mixture of medium - risk and high - risk cases . improved locoregional tumor control has been shown but so far no influence on survival [ 18 ] . from our country a randomized study has presented data for a pure medium - risk group that did include neither low - risk cases nor high - risk cases . the low - risk and high - risk groups have been studied separately in two other randomized protocols , and the results have been presented elsewhere [ 7 , 8 ] . the aim of the present study was to evaluate the prognostic value of the various clinical and histopathological factors commonly discussed in endometrial cancer and how to combine them into risk group definitions . proposals of pre- and postoperative risk groups are presented and tested in a large series of endometrial carcinomas comprising more than 4,500 patients . the only large study published before analyzing prognostic factors in endometrial cancer was the registry seer study , where 41,120 cases were included . figo stage , type of histology , figo grade , lymph node status , age at diagnosis , and race were found to be prognostic factors in that study . the 5-year actuarial locoregional recurrence rate was 3.6% , and the distant recurrence rate was 6.6% in our series . eight of twelve commonly used prognostic and predictive factors were analyzed in this study . with regard to recurrences figo grade , dna ploidy , and depth of myometrial infiltration were independent and significant predictive factors in a multivariate logistic regression analysis . a best subset analysis also confirmed that these three factors were the most important ones , and addition of further factors only marginally improved the predictive value of the model . the single most important factor was the figo grade , but it is important to point out that the dna ploidy , not so commonly used in the international literature , was one of the three most important predictive factors together with myometrial invasion to predict the risk of tumor recurrences , and especially distant recurrences . a number of studies from sweden [ 1417 ] have pointed out the prognostic importance of dna ploidy before , but this information does not seem to have been generally accepted and spread worldwide [ 25 ] . the 5-year actuarial overall survival rate in this series of patients was 73% , and the cancer - specific survival rate was 83% . the study covers a long time period , but in fact the overall survival did not change during the last three decades . changes in the treatment technique during these years seem to have had no impact on survival . cox proportional multivariate regression analysis was used to find out the most important prognostic factors with regard to the cancer - specific survival probability . eight factors were included in the model , and seven were found to be independent and significant . of the included factors only p53 expression was nonsignificant in these analyses . advanced versus early tumor stage was the single most important factor with a risk ratio of 4.2 , and figo grade was the second most important with a risk ratio of 2.5 . interesting findings were that the nuclear grade [ 14 , 15 ] was significant and independent of the figo grade , and the dna ploidy with a risk ratio of 1.6 was more important than myometrial invasion with a risk ratio of 1.3 . in fact , myometrial invasion had the lowest risk ratio of all seven analyzed and significant prognostic factors . tumor size and lymphovascular space invasion ( lvsi ) were not included in the present analyses , since these variables were not regularly reported by the departments of pathology during the extensive study period . tumor size with a cutoff level of 2 cm has been reported to be an important predictive factor in preoperative risk classification to define a low - risk group where lymph node dissection is not required . in another study tumor size lvsi has been pointed out as an important predictive factor associated with lymph node metastases and distant tumor spread [ 2022 ] . three risk groups were analyzed with the definitions used in our country during the last 20 years and also in three published randomized multicenter studies [ 79 ] . interesting to note is that 22% of all tumors belonged to a high - risk group with these definitions and 54% belonged to a low - risk group . the prognoses of the three groups are highly significantly different , and especially the high - risk group showed a poor prognosis with only 50% cancer - specific survival rate . these definitions and risk groups seemed to work out well to discriminate between patients , where surgery alone is enough ( low - risk cases ) , where vaginal brachytherapy should be added ( medium - risk cases ) , and where external beam radiotherapy and chemotherapy probably are the treatment options [ 79 ] . for preoperative risk group classification it is more convenient to use two risk groups . the aim of this classification is to sort out those patients requiring lymph node dissection from those who do not . myometrial invasion is an important predictive and prognostic factor but difficult to assess preoperatively in a reliable way . our multivariate analyses of this large series of patients have shown that myometrial invasion can be replaced by other prognostic factors without losing to much of prognostic information . the results from our analysis showed that histology ( nonendometrioid versus endometrioid ) , figo grade ( grade 3 versus grade 1 - 2 ) , and dna ploidy ( nondiploid versus diploid ) could be used to define two preoperative risk groups . these two risk groups discriminated well ( p < 0.000001 ) between low - risk and high - risk cases with a 30% difference in 5-year cancer - specific survival rate . using this definition , the preoperative high - risk group includes 27% of all new cases of endometrial cancer . in an italian study , preoperative risk classification was made using histology , tumor grade , myometrial invasion , cervical spread , and abdominal spread and correctly identified the postoperative risk classification in 96% with high sensitivity and specificity . the importance of dna ploidy as a predictive and prognostic factor in endometrial carcinoma [ 1417 , 24 ] and part of risk group classifications [ 79 ] is one of the most important results of this study . it is important to analyze large samples of endometrial carcinomas to sort out the most important and significant predictive and prognostic factors that should be used in future risk group classifications . it is also important for coming randomized studies that there will be an international consensus regarding the definition criteria to be used for the various risk groups . risk group definitions are important in the design of randomized studies in endometrial carcinomas . up to now some confusion exists in these definitions in published randomized studies making firm conclusions and comparisons difficult . three risk groups seem reasonable to use in the postoperative setting , but probably only two in the preoperative classification . our study has shown that dna ploidy is an important predictive and prognostic factor and if used in combination with the figo grade and type of histopathology can replace myometrial invasion in definition of preoperative high - risk cases needing more extensive surgery .

background : single - nucleotide polymorphisms ( snp ) in the inosine triphosphate pyrophosphatase ( itpa ) gene correlate with ribavirin ( rbv)-induced anemia in patients with chronic hepatitis c ( chc ) receiving combination therapy . managing anemia is an early priority in the treatment process.objectives:the aim was to develop a predictive index based on itpa snp status to identify chc patients at risk of anemia.patients and methods : a total of 418 eligible east asian patients diagnosed with chc genotype 1 ( g1 ) received combination therapy in this study . participant dna was genotyped for a functional itpa snp ( c / c , a / a or c / a ) on chromosome 20 at rs1127354 . a predictive index was constructed by incorporating independent factors identified for severe anemia events ( hemoglobin < 10 g / dl ) . areas under the receiver - operating characteristic curves ( aucs ) represented the diagnostic accuracies of the predictive index in randomly assigned development and validation cohorts.results:multiple logistic regressions identified age ( 50 y : or = 9.7 , 95% ci = 5.0 - 18.6 ) , itpa rs1127354 ( c / c : or = 3.3 , 95% ci = 1.8 - 5.8 ) and baseline hemoglobin ( < 14.0 g / dl : or 6.4 , 95% ci = 3.3 - 12.1 ; 14.0 - 14.9 : or = 2.4 , 95% ci = 1.2 - 4.6 ) as predictors of severe anemia throughout the treatment . for severe anemia , the predictive index incorporating age , itpa snp status and baseline hemoglobin yielded diagnostic accuracies ( aucs ) of 0.830 ( 95% ci = 0.783 - 0.871 ) in the development ( n = 324 ) and 0.902 ( 0.826 - 0.925 ) in the validation ( n = 81 ) cohorts.conclusions:in patients with chc g1 and receiving combination therapy , itpa snp - based index was an accurate and practical solution for prediction of severe anemia . background . the aim was to evaluate predictive and prognostic factors in a large consecutive series of endometrial carcinomas and to discuss pre- and postoperative risk groups based on these factors . material and methods . in a consecutive series of 4,543 endometrial carcinomas predictive and prognostic factors were analyzed with regard to recurrence rate and survival . the patients were treated with primary surgery and adjuvant radiotherapy . two preoperative and three postoperative risk groups were defined . dna ploidy was included in the definitions . eight predictive or prognostic factors were used in multivariate analyses . results . the overall recurrence rate of the complete series was 11.4% . median time to relapse was 19.7 months . in a multivariate logistic regression analysis , figo grade , myometrial infiltration , and dna ploidy were independent and statistically predictive factors with regard to recurrence rate . the 5-year overall survival rate was 73% . tumor stage was the single most important factor with figo grade on the second place . dna ploidy was also a significant prognostic factor . in the preoperative risk group definitions three factors were used : histology , figo grade , and dna ploidy . conclusions . dna ploidy was an important and significant predictive and prognostic factor and should be used both in preoperative and postoperative risk group definitions .

fistula - in - ano is an abnormal hollow tract lined by unhealthy granulation tissue connecting a primary opening in the anal canal to a secondary opening in the perianal skin . the fistula tract may be single or multiple and may extend from the same primary opening . it is nearly always caused by a previous anorectal abscess , following either poor surgical or spontaneous drainage in the perianal skin . men are involved in 70% of cases and the majority of cases present initially in the 3 to the 6 decade , which incidentally coincide with the mean age occurrence of fournier 's gangrene . fournier 's gangrene , which was first described by jean alfred fournier in 1883 , is an infective necrotizing fasciitis of the perineal , genital or perianal regions as a result of subcutaneous vascular thrombosis and resulting in gangrene of the overlying scrotal skin . fistula - in - ano and fournier 's gangrene still continue to pose management challenges to surgeons . however , there has been no report on management of this concomitant occurrence of these diseases . this is a retrospective study of all fistulas - in - ano complicated by fournier 's gangrene managed in university of maiduguri teaching hospital and federal medical center yola and gombe within a period of 5 years ( january 2007 to december 2011 ) highlighting the causal relationship . the case files of all patients with the diagnosis of fistula - in - ano complicated by fournier 's gangrene during the study period were retrieved from the central medical records department of these hospitals . information retrieved includes the demographic data , mode of presentation , type of infecting organisms , co - morbidity illnesses , nature and outcome of treatment . data analysis was carried out using the statistical package for the social sciences version 16 ( spss 16 ) to determine the level of significance and correlations with the variables . a total of 10 male patients who developed fistula - in - ano and then complicated by fournier 's gangrene with a mean age of 50.5 ( 50 - 59 ) years were managed within the period of 5 years [ table 1 ] . nearly , 50% of patients were peasant farmers , 30% businessmen and 20% were civil servant . all patients presented with initial perianal pain , external opening and discharge and then followed by scrotal swelling and high grade fever [ table 2 ] . nearly , 70% of these patients presented with fournier 's gangrene within 1 - 4 weeks of development of fistula - in - ano , which were preceded by perianal abscess and the rest within 8 weeks [ table 3 ] . fistula - in - ano was submuscular ( intersphincteric ) , 30% subcutaneous with straight anterior external opening and 10% were complex or recurrent running curved course from internal posterior to anterior external opening [ figure 1 ] . age distribution of 10 patients with fistula - in - ano and fournier 's gangrene major clinical features of 10 patients with fistula - in - ano and fournier 's gangrene duration of symptoms prior to presentation of 10 patients with fistula - in - ano and fournier 's gangrene types of fistula - in - ano with fournier 's gangrene at presentation of the 10 patients nearly , 40% of these patients had initial nick for drainage of their perianal abscess and 20% incision and drainage ( i and d ) , but 40% came with spontaneously ruptured perianal abscess , which developed fistula - in - ano and then fournier 's gangrene with no initial treatment [ table 4 ] . types of treatment prior to presentation of 10 patients with fistula - in - ano and fournier 's gangrene all patients were given adequate intravenous fluid and parenteral antibiotics before definitive treatment . 20% of patients had fistulotomy and seton ( rubber band ) application for adequate drainage . in 50% of patients mucosal advancement flap was done to close the internal opening of the fistula , but 30% had fistulotomy and sitz bath [ table 5 ] . types of procedure and surgery for 10 patients with fistula - in - ano and fournier 's gangrene there is a significant relationship between initial treatments , type of fistula - in - ano , durations of the clinical features type of definitive surgical procedure carried out and the outcome [ table 6 ] . the paired t samples tests of association of durations and the clinical features with other variables if the cryptoglandular abscess is either poorly drained or spontaneously ruptures through the perianal skin , then becomes fistula in 50% of patients . severe polymicrobial synergistic infection from the gut flora can cause an extensive subfascial tissue necrosis involving the scrotum , fournier 's gangrene [ figure 2 ] , which is an unusual complication of fistula - in - ano . other common causes of anal fistula include chronic ulcerative colitis , crohn 's disease , tuberculosis , carcinoma of the rectum or anal canal , benign rectal strictures , foreign bodies or diverticulitis . cryptoglandular fistula with fournier 's gangrene fournier 's gangrene , which was thought to be idiopathic by jean alfred fournier ( 1883 ) is now known to have definite etiology and is characterized by obliterative endarteritis causing cutaneous and subcutaneous vascular thrombosis and tissue necrosis , then invaded by polymicrobial enterobacteria . in our region , staphylococcus and proteus species are the commonest mixed growth infection and quinolones ( ciprofloxacin and ofloxacin ) are the most potent antibiotics in treating these infection . urogenital foci such as urethral stricture , indwelling urethral catheter , prostatic message and biopsy are the most common primary adjacent focus of fournier 's gangrene and in africa immunosuppressive illnesses such as diabetes mellitus , human immunodeficiency virus infection , filarial infestations and low socio - economic status contributed to the high prevalence of the disease . in these patients , 50% are in the low socio - economic category , however , 80% of them were otherwise healthy without immunosuppressive illnesses . usually , fournier 's gangrene is presented in the acute form , but recently it runs an indolent course , which some studies attributed it to immunosuppressive comorbidities ; however in our series , it 's probably due to suboptimal antibiotic therapy and delay in referral . these patients presented with high grade fever associated with rigors and sweating , some of them delirious . nearly , 60% of the patients either had a perianal nick [ figure 3 ] or inadequate i and d [ figure 4 ] and 40% had a prior history of spontaneous rupture and discharge from a perianal abscess with worse clinical symptoms . nick of perianal abscess and fournier 's gangrene inadequate perineal abscess drainage and fournier 's gangrene the parks classification system divided fistula - in - ano resulting from cryptoglandular infections as intersphincteric , transsphincteric , suprasphincteric and extrasphincteric , however , the current procedural terminology codes classification include subcutaneous fistula resulting from unhealed anal fissure or anorectal procedures such as hemorrhoidectomy , submuscular fistula ( intersphincteric , low transsphincteric ) and complex fistular , recurrent ( high transsphincteric , suprasphincteric and extrasphinteric , multiple tracts , recurrent ) . majority of fistula - in - ano can be diagnosed based on clinical history and physical examination , however , in cases associated with multiple or complex problems such as a horse shoe abscesses or recurrent complex fistula - in - ano , rectal cancer or crohn 's disease , diagnostic imaging such as transrectal ultrasound , computed tomography scan or magnetic resonance imaging would be required . the parenteral antimicrobials ciprofloxacin and metronidazole were given based on the sensitivity pattern of the microbial pathogens in our region . some of the patients had extensive debridement from the perianal region up to the scrotum and groins leaving the testes bare [ figure 5 ] . hypertonic saline soaking ( sitz bath ) and wound dressing were continued mornings and evenings until the wounds were cleaned and granulated in 2 - 4 months [ figures 6 and 7 ] . if patient presented early with cryptoglandular infection and perianal abscess , the abscess is immediately drained by either radial perianal incision or adequate cruciate incision with unroofing the abscess cavity . these procedures allow adequate abscess drainage and are performed close to anal verge in order to allow fast healing . it is rare for fistula to heal spontaneously even with antibiotic therapy . in order to heal , the internal opening ( crypt ) should be obliterated and therefore operative procedure is necessary . if the internal opening identified , the tract should be probed to clarify and a seton suture of silk passed through to serve as a drain until definitive treatment can be done . nearly , 40% of these patients had a nick of the abscess cavity as primary treatment , which was inadequate i and d and 40% had spontaneous rupture of the abscess cavity . these made perineal spread of the infection rapid and thus prolonged period of patient 's management and increased risk of mortality . although 20% of the patients had adequate i and d , but yet developed a complication of fournier 's gangrene , probably would have benefited from closure of the internal opening as part of primary treatment . extensive perineal and scrotal debridement with bare testes cleaned granulated scrotal and perineal wound ready for grafting healing anal fistula and scrotal wound with continuing sitz bath the operative goals for subcutaneous and submuscular fistulae are to open the tract and remove tract lining either by curettage or electrocautery . the complex or recurrent fistulae may be treated by either non - cutting setons , fibrin glue , ablation , rectal mucosal advancement flap , anal fistula plugs or combination of these in order to avoid sphincter muscle division and its attendant fecal incontinence complication . fistulotomy is a preferred procedure for subcutaneous and submuscular fistulae whether complicated or not and has 3 - 5% risk of flatus and stool leakage and with more muscle cutting fecal incontinence may develop . majority of the patients had their scrotal wounds healed spontaneously with continuing sitz bath [ figure 7 ] , but those with more extensive perineal wound had mesh skin grafting [ figure 8 ] . patients were followed - up over a year with no history of fecal incontinence or fistula recurrence . cryptoglandular infection is an important cause of perianal abscess and fistula - in - ano when poorly managed can lead to fournier 's gangrene , which is a urological emergency . early adequate parenteral antibiotic therapy , primary surgical treatment including closure of internal opening ( crypt ) can prevent serious complications like fournier 's gangrene . congenital coarctation of the aorta is a narrowing of the descending aorta which typically is located at the ligamentum arteriosum just distal to the left subclavian artery . this condition may be undiagnosed until adult life , when the clinical presentation most often is high blood pressure ( bp ) in both or more seldom in only one of the upper extremities . other typical clinical manifestations may include headache , fatigue on exertion , and bilateral lower limb claudication . coarctation of the aorta occurs in 5 - 8% of cases of congenital heart defects . this condition may occur along with ventricular septal defect and other related heart defects , or may occur isolated . in rare cases , severe trauma and injury may lead to coarctation of the aorta . in extremely rare cases , severe atherosclerosis or inflammatory diseases of the aorta may cause narrowing of the artery leading to aortic coarctation . a 57-year - old patient was referred to our outpatient clinic by his primary care physician because the 12-lead ecg demonstrated left ventricular ( lv ) hypertrophy . twenty - two years earlier , the patient had been referred for cardiological examination due to a cardiac systolic murmur . at that time , his bp was 98/50 mmhg , and simultaneous and equal radial and femoral pulses were described . no medical or cardiovascular history or cardiovascular risk factors were present , and the patient had no signs of genetic disorders . at the present consultation , the patient confirmed the absence of any cardiovascular symptoms . transthoracic echocardiography showed a non - dilated , hypertrophic left ventricle [ figure 1a and b ] with end - diastolic interventricular septal thickness of 21 mm , end - diastolic lv posterior wall thickness of 12 mm , and an estimated lv mass of 449 g ( lv mass index 214 g / m ) . the lv ejection fraction was 50% . except for a mild aortic regurgitation ( in a normally shaped tricuspid aortic valve ) and a dilatation of the ascending aorta of 40 mm a continuous wave doppler examination from the suprasternal notch showed a peak systolic pressure gradient in the thoracic descending aorta of 80 mmhg without diastolic run - off [ figure 1c and d ] , indicating a severe obstruction at the classical site of a coarctation . multislice computed tomographic ( ct ) angiography confirmed the finding of severe coarctation of the aorta . the ct scan demonstrated that both subclavian arteries originated distal to the severe coarctation , explaining the normal bp in both arms [ figure 2 ] . moreover , a ct scan of the cerebrum revealed the vessels in the circle of willis giving rise to numerous collaterals in the brain circulation . 57-year - old male was referred to our outpatient clinic because the 12-lead ecg demonstrated left ventricular ( lv ) hypertrophy that was later diagnosed as due to congenital coarctation of the aorta . transthoracic echocardiography ( a ) apical four - chamber view and ( b ) m - mode show left ventricle hypertrophy ( arrows ) ; ( c and d ) suprasternal views show the narrowing in the thoracic descending aorta ( arrow ) and the continuous wave doppler curve without diastolic run - off ( arrow ) . 57-year - old male was referred to our outpatient clinic because the 12-lead ecg demonstrated left ventricular ( lv ) hypertrophy that was later diagnosed as due to congenital coarctation of the aorta . we report an uncommon case of congenital coarctation in a 57-year - old man without the clinical signs of coarctation . because of the uncommon location of the aortic narrowing with both the right and left subclavian arteries originating distal to the area of coarctation , the bp was equally low in both upper extremities . the present case shows that a normal brachial bp does not rule out severe coarctation and should be considered in apparently normotensive patients presenting with a systolic murmur or target organ damage , in this case severe lv hypertrophy . uncorrected coarctation of the aorta in adults predisposes to congestive heart failure , aortic dissection and rupture , stroke , cerebral hemorrhage , and infective endocarditis . therefore , an early diagnosis is important , and the present case emphasizes the use of suprasternal view as a part of a standard diagnostic echocardiography . treatment options include surgical repair or balloon angioplasty with or without stent implantation . taking the atypical location , extent , and complexity of the lesion into account ,

background : fistula - in - ano when complicated by fournier 's gangrene is an unusual finding and always carries high morbidity . this study details our experience in managing 10 cases.methods of study : case files of all patients managed in university of maiduguri teaching hospital and federal medical center of yola and gombe from january , 2007 to december , 2011 were retrieved from medical record departments and other hospital records . these were analyzed for demographic , clinical and pathological variables , the type of treatment and follow-up.results:a total of 10 men with a mean age of 50.5 years ( 35 - 60 ) were managed in the period of study . nearly , 50% of the patients were farmers , 30% businessmen and 20% were civil servant . 7 ( 70% ) of these patients presented with fournier 's gangrene within 4 weeks of development of fistula - in - ano and the rest within 8 weeks . 4 ( 40% ) of these patients had inadequate drainage of their perianal abscess and 2 ( 20% ) had incision and drainage . another 4 ( 40% ) had spontaneously rupture of the perianal abscess . 6 ( 60% ) of the fistula - in - ano was submuscular , 30% subcutaneous and 10% were complex or recurrent . nearly , 20% of patients had fistulotomy and seton application for adequate drainage . mucosal advancement flap was performed in 5 ( 50% ) and fistulotomy in 3 ( 30% ) patients . another 30% had fistulotomy and continuing sitz bath.conclusion:cryptoglandular infection is an important cause of perianal abscesses and fistula - in - ano and if poorly managed results in fournier 's gangrene . early broad spectrum parenteral antibiotic therapy and primary surgical treatment can prevent fournier 's gangrene . the present case shows that a normal brachial blood pressure ( bp ) does not exclude severe coarctation and should be considered in normotensive patients presenting with a systolic murmur and/or unexplained severe left ventricular hypertrophy . congenital coarctation of the aorta is a narrowing of the descending aorta , usually located distal to the origin of the subclavian artery , causing hypertension in the upper part of the body . this condition may be undiagnosed until adult life where the clinical presentation most often is high bp in the upper extremities . a 57-year - old patient with severe aortic coarctation and left ventricular hypertrophy presented with normal brachial bp . however , standard suprasternal view by echocardiography indicated coarctation . multislice computed tomographic ( ct ) angiography revealed an uncommon location of the aortic narrowing with the right and left subclavian arteries originating below the area of coarctation , explaining the equally low bp in both upper extremities .

multistep skin carcinogenesis assays were performed with mice with keratinocyte - specific deletion of the cnb1 gene ( cnb1xk5-crepr1)3 in parallel with cre - negative controls ( cnb1 ) . detailed conditions for these assays as well as chromatin immunoprecipitation , immunoblotting , immunofluorescence , senescence -galactosidase staining , biotinylated dna pull down assays , and sorting can be found in the method section and supplementary figure legends . micrornas ( mirnas ) are single stranded non - coding rna molecules of 22 nucleotides ( nt ) that regulate gene expression via post - transcriptional and/or translational repression ( ambros , 2004 ) . primary mirna ( pri - mirnas ) are transcribed in the nucleus by rna polymerase ii or iii , where 70 nt stem - loop mirna precursors ( pre - mirnas ) are subsequently excised by the microprocessor complex containing the rnase iii enzyme drosha , and exported to the cytoplasm via exportin-5 ( kim et al . , 2009 ) . dicer , another rnase iii enzyme , further processes pre - mirnas to produce mature mirnas , the final product being a 22 base - pair duplex with 2 nt - long 3 overhangs ( he and hannon , 2004 ) . then , one strand of the mature mirna is loaded onto argonaute ( ago , also known as eif2c ) , a core protein of the rna - induced silencing complex ( risc ) . mirna forms base pairs with a target mrna as a guide for ago binding and to direct the specificity of the risc effector , decreasing target mrna levels and/or its translation ( fabian et al . , 2010 ) , where mrna destabilization is the dominant mechanism ( eichhorn et al . , 2014 ; guo et al . , mirnas are abundant in the mammalian genome ( more than 2000 human mirnas are currently reported in mirbase ) ( kozomara and griffiths - jones , 2014 ) and their regulatory role is essential , affecting various biological phenomena ( kim , 2005 ; sim et al . , 2014 ) . supporting evidence derives from the fact that a lethal phenotype during early development was observed in dicer1-null ( bernstein et al . , 2003 ) or ago2-null ( liu et al . , 2004 ) mice , and various biological defects were also reported after losses of individual mirnas ( park et al . , 2010 ) . in addition , alterations of mirna regulation are related to many diseases such as neurological disorders ( hebert and de strooper , 2009 ) , various types of cancer ( croce , 2009 ) , and cardiovascular diseases ( olson , 2014 ) . importantly , all of these defects were ultimately caused by a dysregulation in target gene expression . however , the limitation here is our ability to delineate a general principle for identification of specific rna targets upon which mirnas act . the problem stems from the observation that most of mirna target sites have partial complementarity ( ambros , 2004 ) . in contrast to plants , a near - perfect base pairing of mirna to its target is rare in animals , making it a challenge to predict the target sites ( bartel , 2009 ) . however , initial prediction attempts provided evidence that local short stretches ( 6 nt ) of consecutive base - pairing significantly contribute to target recognition ( john et al . , 2004 ; krek et al . , 2005 ; lewis et al . conceptually termed as the nucleus , the short consecutive matches could initiate a mirna - target duplex , followed by the propagation of partial annealing that may further stabilize mirna - target hybridization ( filipowicz , 2005 ; rajewsky , 2006 ) . intriguingly , nuclei were further found to be typically located in the 5 end region of mirnas called the seed , enabling the prediction of mirna target sites ( lewis et al . , functional mirna - target interactions are known to majorly require as few as 6-nt matches within the seed region ( position 2 - 8 , fig . there are possible 6-mers ( positions 16 , 27 , and 38 ) , 7-mers ( positions 28 and 17 ) , and 8-mer ( position 18 ) matches in the seed . otherwise , offset 6-mer seed because of its position and a marginal effect on repression ( friedman et al . , 2009 ) . such canonical seed sites were initially known by early biological studies ( lee et al . , 1993 ; poy et al . , 2004 ; wightman et al . , 1993 ) , which were further validated by microarray experiments that detected enrichment of seed matches in mirna - dependent transcripts showing repression ( grimson , 2007 ; lim , 2005 ) , and also by bioinformatics analyses , which found widespread conservation of seed sites in 3 untranslated regions ( 3 utrs ) in multi - genome sequences ( lewis et al . , 2005 ; xie et al . , 2005 ) . seed - pairing rules have been informative in prediction and analysis of canonical seed sites , often in combination with evolutionary conservation ( friedman et al . , 2009 ; lewis et al . , 2005 ) , secondary structure ( long et al . , however , since a 6 nt match presents on average every 4,000 nt , likely to be occurred often by chance , such strategies still suffer from both false - positive ( 4066% ) and false - negative predictions ( 5070% ) ( mourelatos , 2008 ) even in the usage of microarray or proteomic approaches ( baek et al . , seed - pairing rules can not identify non - canonical target sites , which have been reported as functional ( brodersen and voinnet , 2009 ) . since seed - pairing rules are widely adopted , there has been an unintentional bias to study only the canonical seed matches , overlooking the non - canonical targets . nevertheless , several biological studies have functionally validated that perfectly matched mirna seeds are neither necessary nor sufficient for all functional mirna - target interactions ( brodersen and voinnet , 2009 ) . for example , supplementary components in near - perfect sites compensate for imperfect seed matches and are functional for target cleavage ( mir-196 for hoxb8 , fig . 1b ) as well as the wobble ( g : u pairing ) in seed sites of lin-41 with 3 compensatory pairings ( vella et al . , lsy-6 can also tolerate wobble paring in the seed to downregulate its target , cog-1 ( didiano and hobert , 2006 ) . in mammals , nanog , oct4 and sox2 genes , well - known regulators generating induced pluripotent stem ( ips ) cells , have been shown to contain functional wobble ( mir-296 for nanog , fig . 1b ) and bulge pairing sites for cognate mirna seeds ( mir-134 , mir-296 , and mir-470 ) , harboring a few cases in their coding sequences ( cds ) ( tay et al . , in contrast to a fruit fly genetic study where 3 compensatory sites were shown to be as functional as the 5 dominant canonical seed sites ( brennecke et al . , 2005 ) , such non - canonical mirna target sites were revealed to be rare in mammals ( less than 5% ) ( friedman et al . , 2009 ) and their effects were modest ( grimson , 2007 ; wee et al . , 2012 ) . when seed - pairing rules were applied , putative mirna targets from microarrays that showed mirna - dependent repression were often demonstrated to have high false - negatives , implicating prevalent usage of non - canonical target sites . in lieu of this , microarray analysis of mir-24-transfected k562 cells found that several mir-24 targets are repressed through non - canonical sites , named seedless 1b ) ( lal et al . , 2009 ) . in addition , centered sites , comprising 1112 consecutive base - pairing to the center of mirna , were also identified by the analysis of microarray data where neither perfect seed nor 3 compensatory pairing was observed ( fig . however , the limitation of such studies is that , lacking information on precise binding sites , they are unable to distinguish between direct and indirect mirna targets . uncertainty in direct mirna target sites necessitates the development of experimental methods capable of recovering mirnas physically associated with their targets ( easow et al . , 2007 ) . initially , biochemical isolation of mirna - mrna complexes via ago protein - specific immunoprecipitation was attempted in order to purify mrnas bound by ago - mirna ( easow et al . , 2007 ; hammell , 2008 ; hendrickson et al . , 2008 ; karginov , 2007 ) . however , the integrity of the approach was questioned because of the possible high background caused by nonspecific rna - protein interactions , especially mediated by in vitro rearrangements in the ago - rna complex ( mili and steitz , 2004 ; riley et al . , 2012b ) . however , the cross - linking and immunoprecipitation ( clip ) method that uses ultraviolet ( uv ) irradiation to crosslink rna - protein complexes in direct contact within approximately single angstrom distances in living cells , can secure specific rna - protein interactions by allowing stringent purification ( ule , 2003 ) . combined with high - throughput sequencing ( hits - clip ) ( licatalosi , 2008 ) , it was successfully applied to ago ( ago hits - clip ) to produce transcriptome - wide information of mirna binding sites ( chi et al . , 2009 ) . the novelty lies not only in providing direct mirna target sites , but also in accomplishing high target specificity ( 93% ) , low false - positives ( 1327% ) , and low false - negatives ( 1525% ) ( chi et al . , furthermore , by analyzing crosslinking - induced mutation sites ( cims ) , footprints of ago - mirna binding regions ( 4562 nt ) became available at single nucleotide resolution ( zhang and darnell , 2011 ) . a modification of this method , par - clip ( photoactivatable - ribonucleoside - enhanced crosslinking and immuno - precipitation ) , used a nucleoside analog that improved the efficiency of uv crosslinking and subsequent mutations ( hafner et al . , 2010 ) , and iclip ( individual - nucleotide resolution uv crosslinking and immunoprecipitation ) analyzed truncated cdnas at the position of the protein - rna crosslink sites ( konig et al . , 2010 ) . ago hits - clip was the first to offer a general means of mapping precise mirna target sites and has been widely applied to cultured cells ( haecker et al . , 2012 ; hafner et al . , 2010 ; kim et al . , 2015 ; kishore et al . , 2011 ; leung et al . ; loeb et al . , 2012 ; riley et al . , 2012a ; xue et al . , 2013 ) , tissues ( boudreau et al . , 2014 ; chi et al . , 2009 ; kameswaran et al . , 2014 ) , and even to a whole organism ( c. elegans ) ( grosswendt et al . , 2014 ; zisoulis et al . , 2010 ) it also provides an opportunity to analyze general properties of mirna - target interactions in an unbiased and transcriptome - wide manner . intriguingly , ago hits - clip analysis revealed that not all identified direct ago - target interactions follow canonical seed - pairing rules ( chi et al . indeed , seed matches for 90% of ago - bound mirnas only explain 73% of ago - mrna interactions in the mouse brain , and the rest of 27% are orphans with no predicted seed matches , strongly suggesting that non - canonical mirna target sites might be prevalent in vivo . non - canonical mirna - target sites called nucleation bulges were identified by analyzing ago hits - clip orphan clusters ( chi et al . , 2012 ) . initially , g - bulge sites for mir-124 were found to be abundant in the mouse brain , where the target sites matched to the seed ( positions 27 ) contained a bulged - out g nucleotide corresponding to position between 5 and 6 of the mirna ( mir-124 for mink1 , fig . upon further analysis , a general rule governing such non - canonical interactions , called the pivot pairing rule , was proposed ( fig . this rule dictates that the nucleotide composition in the bulge position should be determined by the base - pairing competency to a nucleotide in position 6 of the mirna , named pivot to confer the thermodynamic stability on the consecutive 5 base pairs of nucleation ( termed nucleation bulge ; figs . otherwise , the base - pair - mediated interaction of only the 4 nt resulting from the non - competent nucleotide in the pivot ( non - nucleation bulge ) is unstable and not functional . well accommodates a canonical interaction mode through nucleation pairing in position 26 , which is followed by the propagation to position 68 of the mirna ( fig . application of the pivot pairing rule successfully decoded the non - canonical nucleation bulge sites , comprising 15% of all ago - mirna - mrna interactions in the mouse brain ( chi et al . , nucleation bulge sites were also observed in ago hits - clip analyses performed in the human brain ( boudreau et al . , 2014 ) and several cell lines ( hafner et al . , 2010 ) . in addition , their sequences are evolutionally conserved ( chi et al . , 2012 ) . the pivot pairing rule improved both quality and quantity of mirna target sites in their identification ( stefani and slack , 2012 ) since it can serve as a general rule that can be incorporated in any computational analysis ( kim et al . , 2013 ) . in addition to nucleation bulges , seed - like motifs that contain mismatches in seed pairing were found by examining differential ago hits - clip binding sites in mir-155 deficient t cells ( mir-155 for gimap3 , fig . 1c ) , indicating that 20% of ago - mir-155 binding sites contain seed - like motifs ( loeb et al . , 2012 ) . recent variants of ago hits - clip , which can directly sequence the binding sites together with mirnas by inducing ligation between mirnas and their target rna sites , also confirmed the widespread occurrence of non - canonical seed - like motifs including some nucleation bulges initially , clash ( cross - linking , ligation , and sequencing of hybrids ) identified that 60% of seed interactions are seed - like motifs in hek293 t cells ( helwak et al . , soon after , modified par - clip and analyses of mirna - target chimeras in ago hits - clip showed that 30% of mirna - target chimeras were also seed - like motifs more recently , clear - clip ( covalent ligation of endogenous argonaute - bound rnas ) also reported the same observation ( moore et al . , 2015 ) . although most of the non - canonical interactions were identified by ago hits - clip , it would be difficult to explain their occurrence through globally applicable rules . however , non - canonical nucleation bulges could be governed by a general rule of pivot pairing , leading to further insights into mirna - target recognition within other similar transcriptome - wide non - canonical interactions . 2 ) ( chi et al . , 2012 ) . combining the concepts of a nucleus ( filipowicz , 2005 ; rajewsky , 2006 ; tomari and zamore , 2005 ) and findings from structural studies for recognition mechanisms of ago silencing complexes - nucleation , propagation and cleavage of target rnas ( schirle et al . , 2014 ; wang et al . , 2009 ) , transitional nucleation is defined as a transient mirna - target duplex with a 5-base - paired nucleation ( position 26 ) ( fig . if the transitional nucleation becomes sufficiently stable to form , this state may be further transformed into a bulge formation where the originally matched pivot nucleotide in position 6 becomes bulged - out and subsequently extended to hybridization towards the 3 end of the mirna ( further than position 6 , fig . this model is also well supported by several structural studies of ago ( elkayam et al . , 2012 ; nakanishi et al . , 2012 ; schirle and macrae , 2012 ) where nucleotides poised for transitional nucleation ( position 26 ) are particularly prearranged becoming a - form helical structures , which are susceptible for base pairing . intriguingly , such a - form - like helical geometry is disrupted after a pivot ( between position 6 and 7 ) formed by a kink resulting from the insertion of the amino acid isoleucine ( i365 ) from the human ago2 protein ( elkayam et al . thus , in theory , any target site pairing to the seed region ( either a seed match or a nucleation bulge ) requires a shift of this nuclear helix to overcome the kink . in support of this model , single - molecule analysis showed such stepwise processes whereby ago2 initially scans for target sites using a small region ( position 24 ) ( chandradoss et al . , 2015 ) and subsequently mediates a rapid and stable binding to the seed region of a mirna ( jo et al . , 2015 ; salomon et al . , 2015 ) , serving as a proofreading procedure for target recognition ( yao et al . , 2015 ) . mirna is reshaped by loading onto ago , being divided into several functional domains - the anchor , seed , central , 3 supplementary , and tail regions ( fig . 3 ) ( salomon et al . , 2015 ; schirle et al . , 2014 ; wee et al . , 2012 ) . importantly , the seed region has two prearranged continuous base stacking configurations ( positions 26 and 79 ) caused by kinks at nucleotides 67 and 910 ( fig . 3a ) ( elkayam et al . , 2012 ; schirle and macrae , 2012 ; schirle et al . , 2014 ) . therefore , transitional nucleation starts pairing through helix 26 and subsequently propagates to helices 79 , overcoming the kink at 67 for cases of 5 dominant interactions ( fig . 3b ) , such as the seed ( bartel , 2009 ) or nucleation bulge sites ( chi et al . , the opposite may also happen for central dominant interactions ( shin et al . , 2010 ) - the interaction could be initiated by paring through helix 79 along with the central region ( positions 1012 ) and further expand up to the 3 supplementary region ( positions 1316 ) ( fig . 3c ) . in this case , crossing the barrier of the kink at position 910 may be required . for seed - like motifs , where seed sites contain mismatches , deletions , or wobble pairings ( grosswendt et al . , 2012 ; moore et al . , 2015 ) , transitional nucleation may require 3 compensatory interactions ( fig . 3d ) , which could be a general determinant of additional specificity for ago binding as shown by clear - clip ( moore et al . , 2015 ) . biological systems initially generate marginally effective non - canonical regulations when existing biological mechanisms require alternative strategies to compensate for what major canonical pathways have been unable to accomplish . followed by this notion , majority of non - canonical mirna - target sites were shown to mediate gene repression at a modest level ( chi et al . , 2012 ; , 2009 ; loeb et al . , 2012 ; moore et al . , 2015 ) only except for centered sites ( shin et al . , 2010 ) , which can trigger slicing activity of ago but only exist as few in whole transcriptome . however , such modest repression , shown by which the most of non - canonical target sites including nucleation bulges for mirnas , was often observed as insignificant in large - scale gene expression analyses ( agarwal et al . , 2015 ) . these were possibly because the marginal repression was confounded by the issue of cellular heterogeneity , variability derived from secondary effects of target repression , and differences in sensitivity and threshold used in the analyses , or non - canonical sites identified by ago clip based methods could be the true interaction but may not be always functional as the consequence of transient bindings for searching targets ( chandradoss et al . , 2015 ) or as requiring combinatorial occurrences of target sites ( krek et al . in fact , evaluation of target repression at individual single cell level ( moore et al . , 2015 ) and gene expression analyses in combination with clip data ( chi et al . , 2012 ; , 2013 ; loeb et al . , 2012 ; moore et al . , 2015 ) did observe significant repression mediated by non - canonical sites albeit the effect is still marginal . since clip data only indicate the direct bindings , they should be analyzed together with gene expression data to access the functionality . future studies should be performed carefully to clear out such issues whenever they analyze marginal effects from non - canonical interactions . the modest effects from the widespread non - canonical sites are likely to be caused by reduced numbers of targets bound by ago - mirna ( chi et al . , 2012 ; grosswendt et al . , 2014 ; , 2012 ; moore et al . , 2015 ) probably due to low binding affinity . in support of this , any mismatches or wobbles in the seed region decrease target binding but enhance the turnover of the risc complex ( wee et al . , 2012 ) , suggesting that non - canonical binding can induce intermediate affinity without affecting the concentration of the ago complex . additionally , this may be a mechanism that provides an unbound ago - mirna complex to adjacent target sites , as shown by the single - molecule analysis where the lateral diffusion from weakly bound ago promoted cooperation between neighboring target sites ( chandradoss et al . , 2015 ) . as a result , combination of canonical and non - canonical sites may provide a variety of spectra in the regulation of gene expression , enabling a fine - tuning of repression activity . moreover , relative to canonical seed sites , non - canonical sites have modest sequence conservation across species ( chi et al . , 2012 ; , 2012 ; moore et al . , 2015 ) , suggesting that they may be evolutionary intermediates under selective pressure for a shift towards high affinity seed sites . in addition , gene ontology analysis of ago hits - clip showed that the majority of non - canonical targets have similar functions to the canonical ones , although they are slightly different in detail ( chi et al . , 2012 ; loeb et al . , 2012 ; this indicates that non - canonical targets may have a different biological function that needs to be acquired to improve or compensate for the canonical targets . although several non - canonical mirna - target sites were reported as functional , they did not receive much intention since they could not be definitely defined ( brodersen and voinnet , 2009 ) . however , ago hits - clip method , which can generate a precise transcriptome - wide map of mirna target sites ( chi et al . , 2009 ) , unexpectedly revealed that large portion of mirna - target interactions are non - canonical ( chi et al . , 2012 ) . advances in ago hits - clip analyses further identified non - canonical nucleation bulges , 2012 ; moore et al . , 2015 ) , expanding our knowledge in the understanding of mirna targets and their functions . moreover , the transitional nucleation model , yielded by the analytic process of explaining the pattern of nucleation bulges , offers a general molecular model that can be used to understand the mechanism of mirna target recognition through seed regions ( chi et al . , 2012 ) . extending this knowledge to applications of rna silencing , modified sirnas that contain abasic substitution in the pivot ( position 6 ) were recently developed to completely eliminate mirna - like off - target repression ( lee et al . , 2015 ; seok et al . , . biological significance of the non - canonical interactions could be interpreted as evolutional intermediates with slightly distinct functions .

calcineurin inhibitors such as cyclosporin a ( csa ) are the mainstay of immunosuppressive treatment for organ transplant recipients . squamous cell carcinoma ( scc ) of the skin is a major complication of treatment with these drugs , with a 65100 fold higher risk than in the normal population1 . by contrast , the incidence of basal cell carcinoma ( bcc ) , the other major keratinocyte - derived tumour of the skin , of melanoma and of internal malignancies increases to a significantly lesser extent 1 . here we report that genetic and pharmacological suppression of calcineurin / nfat function promotes tumour formation in mouse skin and in xenografts , in immune compromised mice , of h - rasv12 expressing primary human keratinocytes or keratinocyte - derived scc cells . calcineurin / nfat inhibition counteracts p53-dependent cancer cell senescence thereby increasing tumourigenic potential . atf3 , a member of the enlarged ap-1 family , is selectively induced by calcineurin / nfat inhibition , both under experimental conditions and in clinically occurring tumours , and increased atf3 expression accounts for suppression of p53-dependent senescence and enhanced tumourigenic potential . thus , intact calcineurin / nfat signalling is critically required for p53 and senescence - associated mechanisms that protect against skin squamous cancer development . micrornas ( mirnas ) are small non - coding rnas ( 22 nucleotides ) regulating gene expression at the post - transcriptional level . by directing the rna - induced silencing complex ( risc ) to bind specific target mrnas , mirna can repress target genes and affect various biological phenotypes . functional mirna target recognition is known to majorly attribute specificity to consecutive pairing with seed region ( position 28 ) of mirna . recent advances in a transcriptome - wide method of mapping mirna binding sites ( ago hits - clip ) elucidated that a large portion of mirna - target interactions in vivo are mediated not only through the canonical seed sites but also via non - canonical sites ( 1580% ) , setting the stage to expand and determine their properties . here we focus on recent findings from transcriptome - wide non - canonical mirna - target interactions , specifically regarding nucleation bulges and seed - like motifs . we also discuss insights from ago hits - clip data alongside structural and biochemical studies , which highlight putative mechanisms of mirna target recognition , and the biological significance of these non - canonical sites mediating marginal repression .

prostate cancer , one of the most commonly diagnosed male malignancies in western countries , is now the leading cause of cancer - related death in men . over the years , genetic epidemiological evidence has accumulated in favor of a considerable hereditary component in prostate cancer susceptibility . genetic linkage analysis in 360 hereditary prostate cancer pedigrees revealed the presence of a hereditary prostate cancer susceptibility gene(s ) at xq27 - 28 ( hpcx ) . in agreement with this finding , further data were obtained by analyzing linkage disequilibrium of molecular markers of x chromosome in a finnish x haplotype . xq27 is a region containing a number of genes important in cancer and embryonic development , including the sperm protein associated with the nucleus in the x chromosome ( spanx ) gene family that consists of the two subfamilies spanx - n and spanx - a / d ( human genome build 37.1 ) . spanx - a / d genes map within segmental duplications , which are regions involved in genomic rearrangements resulting in an abnormally high level of structural polymorphisms . accordingly , the spanx - b and the spanx - c genes were shown to be present in a variable number of copies ( ranging from one to > 11 ) in the normal population ; however , no association was found between spanx copy number and the occurrence of hereditary prostate cancer by the genetic locus described by xu , thus leaving uncertain the possible identification of the aforementioned locus with spanx gene cluster . spanx proteins are normally expressed in germ cells ; however , their expression has also been detected in a number of tumors , including melanoma , myeloma , glyoblastoma , breast carcinoma , prostate cancer , and testicular germ cell tumors . the present study was undertaken to evaluate the expression of spanx proteins in normal prostate tissues and in prostate cancer by immunohistochemistry . the analysis was carried out on 15 normal ( at autopsy ) donors and 12 men with prostate cancer following radical prostatectomy . patients ( 71.71.8 years ) and normal controls ( 74.51.3 years ) had a similar mean age ( table 1 ) . patients ' histological diagnosis , gleason scores and pre - surgery serum psa levels are shown in table 1 . table 1characteristics of healthy prostate tissue donors and of patients who underwent radical prostatectomy for prostate carcinoma.normal prostate ( autopsy)prostate carcinoma ( radical prostatectomy)idage ( years)positive cell nuclei ( % ) positive cell cytoplasm ( % ) idage ( years)psa ( ng / ml)ptngleason scoregpositive cell nuclei ( % ) positive cell cytoplasm ( % ) 0.163001727.8pt304 + 3g280700.277002639.2pt304 + 4g3000.3725503798.7pt3na4 + 4g3000.4716004707.6pt303 + 3g275650.5680057211.6pt3na4 + 3g280600.680006796.8pt2bna3 + 3g2000.7795507728.2pt303 + 2g175600.877008779.6pt2b03 + 4g2000.97650097410.1pt3na4 + 4g2000.10800010739.9pt304 + 3g2000.117160011578.7pt304 + 3g285750.12740012729.5pt2b02 + 2g1000.1379000.1472000.1578450id , identification ; psa , prostate specific antigen ; pt , tumor stage ; n , lymphonodes ; na , lymphonodes not excissed ; g , grading . i d , identification ; psa , prostate specific antigen ; pt , tumor stage ; n , lymphonodes ; na , lymphonodes not excissed ; g , grading . the protocol was approved by the internal institutional review board and an informed written consent was obtained from each patient with prostate cancer or , if deceased , by his relatives . four m formalin - fixed and paraffin - embedded prostate sections were processed following the standard protocol previously described . we did not observe any time - related effect on immunostaining with the antibody used for this study nor with any other antibody used for diagnosis . a polyclonal serum against the common spanx epitope tptgdsdpqp , developed in mouse , was used , as reported in a previous study . as negative control , anti - spanx serum was pre - incubated with the immunizing peptide ( 100 ng ) for 1 h. tissues were visually scored at 20x magnification for spanx positivity ; the fraction of spanx - positive cells was evaluated independently in a blinded fashion by two of us in microscopic fields where non prostatic cells ( infiltrating leukocytes , fibroblasts , etc . ) were as few as possible . since no significant difference was observed between the two observers , a mean value was used . comparisons between the percentages of spanx - positive cells were carried out by the student 's t test ( spss 9.0 software package for windows ) . the analysis was carried out on 15 normal ( at autopsy ) donors and 12 men with prostate cancer following radical prostatectomy . patients ( 71.71.8 years ) and normal controls ( 74.51.3 years ) had a similar mean age ( table 1 ) . patients ' histological diagnosis , gleason scores and pre - surgery serum psa levels are shown in table 1 . table 1characteristics of healthy prostate tissue donors and of patients who underwent radical prostatectomy for prostate carcinoma.normal prostate ( autopsy)prostate carcinoma ( radical prostatectomy)idage ( years)positive cell nuclei ( % ) positive cell cytoplasm ( % ) idage ( years)psa ( ng / ml)ptngleason scoregpositive cell nuclei ( % ) positive cell cytoplasm ( % ) 0.163001727.8pt304 + 3g280700.277002639.2pt304 + 4g3000.3725503798.7pt3na4 + 4g3000.4716004707.6pt303 + 3g275650.5680057211.6pt3na4 + 3g280600.680006796.8pt2bna3 + 3g2000.7795507728.2pt303 + 2g175600.877008779.6pt2b03 + 4g2000.97650097410.1pt3na4 + 4g2000.10800010739.9pt304 + 3g2000.117160011578.7pt304 + 3g285750.12740012729.5pt2b02 + 2g1000.1379000.1472000.1578450id , identification ; psa , prostate specific antigen ; pt , tumor stage ; n , lymphonodes ; na , lymphonodes not excissed ; g , grading . i d , identification ; psa , prostate specific antigen ; pt , tumor stage ; n , lymphonodes ; na , lymphonodes not excissed ; g , grading . the protocol was approved by the internal institutional review board and an informed written consent was obtained from each patient with prostate cancer or , if deceased , by his relatives . four m formalin - fixed and paraffin - embedded prostate sections were processed following the standard protocol previously described . we did not observe any time - related effect on immunostaining with the antibody used for this study nor with any other antibody used for diagnosis . a polyclonal serum against the common spanx epitope tptgdsdpqp , developed in mouse , was used , as reported in a previous study . as negative control , anti - spanx serum was pre - incubated with the immunizing peptide ( 100 ng ) for 1 h. tissues were visually scored at 20x magnification for spanx positivity ; the fraction of spanx - positive cells was evaluated independently in a blinded fashion by two of us in microscopic fields where non prostatic cells ( infiltrating leukocytes , fibroblasts , etc . ) were as few as possible . since no significant difference was observed between the two observers , a mean value was used . comparisons between the percentages of spanx - positive cells were carried out by the student 's t test ( spss 9.0 software package for windows ) . normal tissues showed only a spanx - positive nuclear signal in 6 samples out of the 15 examined ( 40% ) , with a mean of 21.77.2% cells with spanx - positive nuclei ( figure 1 a , c ; table 1 ) . the expression of spanx was also evaluated in 12 samples of prostate carcinoma with a mean gleason score of 3.50.2 and 3.20.2 and a grading ranging from g1 to g3 . five samples exhibited a spanx - positive nuclear signal ( 41.7% ) with a mean of 32.911.8% positive nuclei ( figure 1 b , d ; table 1 ) . moreover , cytoplasmic staining for spanx was observed in the 27.59.9% of the cells ( range : 6075 ) . none of the controls , analyzed in parallel , exhibited cytoplasmic staining ( p<0.001 vs. prostate carcinoma samples ) . no cytoplasmic staining was observed in the absence of nuclear staining in prostate cancer tissue ( table 1 ) . ( a ) normal prostatic tissue ; ( b ) prostate carcinoma ( a , b hematoxylin counterstain ; bar = 800 m ) ; ( c ) normal prostatic tissue ( hematoxylin counterstain ; bar = 200 m ) ; ( d ) prostate carcinoma ( hematoxylin counterstain ; bar = 80 m ) . ( a ) normal prostatic tissue ; ( b ) prostate carcinoma ( a , b hematoxylin counterstain ; bar = 800 m ) ; ( c ) normal prostatic tissue ( hematoxylin counterstain ; bar = 200 m ) ; ( d ) prostate carcinoma ( hematoxylin counterstain ; bar = 80 m ) . this is the first demonstration that spanx genes are expressed in a normal somatic tissue , apart the already known expression in germ cells . the percentage of cells showing a spanx - positive nuclear staining was comparable both in normal and in pathologic tissues ; prostate cancer cells showed also a cytoplasmic staining . this peculiar feature of spanx staining , if confirmed by larger cohort studies , may be of clinical usefulness for the immunohistochemical differential diagnosis of prostate carcinoma . cytoplasmic spanx labeling is a common finding in malignant cancer cells , such as embryonal carcinomas and melanomas . in addition , evidences of cytoplasmic labeling in melanomas has been shown by westbrook et al . on the basis of extensive observations of histological samples , we believe that the cytoplasmic signal represents a leakage of spanx proteins from the nucleus . a definitive answer will require the study of tumor cells lines expressing spanx . it is conceivable that similar events , which have the potential of altering the regulation of gene expression , may also be present in malignant cells . the similar fraction of spanx positive cells in normal and cancer tissues suggests that the expression of spanx genes in prostate carcinomas correlates with their expression in normal cells from which the tumor originates : a finding similar to what previously described in seminomas , where nonetheless the nuclear positivity involves approximately 100% of the cells . since our antibody was generated by immunizing mice with a synthetic peptide , which sequence is shared by almost all known spanx proteins , we can not differentiate spanx - b form spanx - a - like subfamilies . although this is a limiting aspect , we believe that , given the high homology among spanx gene family members , it would be difficult to assess the specificity of any antibody for a spanx subfamily or for a single member , especially for immunohistochemical purposes . in conclusion , the present findings showed that spanx expression is physiological in 40% of normal individuals , at least in the sicilian sample examined in this study . the similar incidence of spanx - positive cells in normal and neoplastic tissues , although suggestive , is not sufficient to establish a causative relationship between spanx expression and prostate cancer tissues , and deserves more detailed studies in additional patients . gli2 mice , in which the bovine k5 promoter drives the constitutive expression of mouse gli26 , develop bcc and bfh6,7 , which are both linked to deregulated hh signaling in humans7,8 . gli2 mice show a reproducible pattern of lesions on the ear that successively involves hyper - keratosis ( flaking ) , thickening and hyperpigmentation ( supplemental fig . mice were scored as positive for the onset of lesions upon the first sign of macroscopic hyperkeratosis in ear tissue . histologically , the lesions present between p80 and p120 resemble bfh as described 7,8 . by p180 , larger , nodular , bcc - like tumors frequently occur deeper in the dermis ( supplemental fig . induction of k17 , a gli target gene9 , is the main alteration in keratin expression prior to onset of lesions in gli2 epidermis ( supplemental fig . gli2 and k17 mice6,10 were interbred so as to assess the impact of k17 loss on genesis of bfh - like tumors . appearance and progression of hamartoma - like lesions were captured from p30 to p125 . at p80 , epithelial lesions are clearly less pronounced in gli2/k17 than in gli2 ear tissue ( fig . in male gli2 and gli2/k17 mice the average onset of lesions is 652 days ( n=32 ) and 912 days ( n=31 ; p < 0.01 ) , respectively . in females , onset is at 805 days ( gli2 ; n=22 ) vs. 1012 days ( gli2/k17 ; n=21 ; p < 0.01)(fig . loss of k17 does not impact gli2 subcellular localization or hedgehog signaling ( supplemental fig . therefore , the absence of k17 causes a delay in the inception of bfh - like skin tumors in gli2 mice . histological anomalies common to hh pathway - activated mouse skin7 were scored in gli2 and gli2/k17 ear tissue ( supplementary fig . 3b ) , are prominent in gli2 ear but markedly reduced in gli2/k17 ear ( supplementary fig . 3c ) . overall tissue thickness and penetration of epithelial downgrowths are also reduced in gli2/k17 ear tissue ( supplementary fig . k17 , k5 , and k14 are uniformly distributed in the lesional epithelium ( supplemental fig . co - assembly of k5 and k17 in gli2 lesional epithelium is conveyed by their co - localization and co - immunoprecipitation ( supplemental fig . the wound - inducible k6 , k6 and k16 , absent in intact epidermis , are induced in the upper layers of thickened gli2 epidermis , preferentially , but are markedly reduced in gli2/k17 skin ( supplemental fig . reduced proliferation , rather than increased cell death , is a key contributor to delayed tumor onset in gli2/k17 skin . relative to gli2 , indeed , the frequency of mitotically - active cells is depressed by > 3-fold in gli2/k17 ear epithelium ( fig . g ) . in contrast , tunel - positive , apoptotic cells are restricted to the upper epidermis of lesional skin and show similar density in both genotypes ( fig . inflammation has emerged as a driver of angiogenesis and tumor growth12 and coincides with k17 induction and loss of barrier function in several skin diseases13,14 . immunoreactivity for markers of innate immune cells ( cd11b ) , t cells ( thy-1 ) , and phagocytes ( inos ) are enhanced in gli2 compared to gli2/k17 ear skin of p80 male mice ( fig . 2a ) , reflecting decreased angiogenesis . myeloperoxidase ( mpo ) enzymatic activity , inherent to neutrophils15 , is increased 17.4 0.5 fold in p80 male gli2 ear tissue but only 5.8 0.1 fold in gli2/k17 males ( data normalized to p80 female gli2 ear ; fig . female gli2/k17 mice also show a reduced level of mpo activity at p80 , being at 0.75 fold of that seen in gli2 controls ( fig . . skin barrier integrity , assessed via a whole - mount dye penetration assay16 , is intact as expected in p70 wildtype ear skin ( fig . in contrast , a sizable portion of the ear is dye - permeable in p70 gli2 mice ( fig . 2c ) ; again , this readout is markedly decreased in gli2/k17 mice ( fig . 2d ) , mpo activity is 5.91.9 fold greater in male gli2 mice relative to females , substantiating the gender bias in this model . mpo activity is lower in p40 male gli2/k17 mice ( 0.55 0.20 relative to female gli2 mice ; fig . 2d ) , mitotic activity is higher in gli2 vs. gli2/k17 epidermis at p40 ( 0.52 0.01 vs. 0.14 0.02 brdu labeled cells / mm of epidermis ) ( fig . barrier integrity is mildy compromised in p40 gli2 ear skin , is again better preserved in gli2/k17 mice ( supplemental fig . thus , the marked reductions in inflammation and hyperplasia that define gli2/k17 ear skin occur as early as p40 , ahead of progression to overt tumorigenesis in the gli2 model . expression of inflammatory cytokines and chemokines was examined via qrt - pcr in ear tissue at p40 and p80 . the findings are stratified according to specific classes of t - helper cytokines : th1 ( cellular immunity ; generally pro - inflammatory ) , th2 ( humoral immunity ; anti - inflammatory ) , and th17 ( anti - microbial immunity at epithelial barriers)17,18 . th1 and absence of k17 in gli2 skin correlates with a marked reduction in th1- and th17-related markers and induction of th2-related markers ( table 1 ) , many of which are prominently expressed by skin keratinocytes themselves . expression of il-1 , a keratinocyte mitogen20 , is ~10 fold higher in gli2 compared to gli2/k17 skin ( table 1 ) . spp1 ( osteopontin ) , which acts to bias immune responses toward th121 , is reduced by ~15 fold and il-6 , associated with the acute phase response and upregulated in human bcc22 , is lowered ~17 fold in gli2/k17 skin ( table1 ) . the matrix metalloproteases mmp3 , mmp9 and mmp13 , whose expression is enhanced in bcc23 , are downregulated in gli2/k17 ear tissue . classical th2 type cytokines primarily secreted by t - cells , e.g. , il-4 and il-10 , are modestly altered whereas ccl24 and ccr4 , expressed by skin keratinocytes24 , are respectively ~9 and ~3 fold higher in gli2/k17 ear tissue ( table 1 ) . il1 and cxcl5 expression is enhanced in the presence of k17 , while the th2 markers il20 and il4 are enhanced in its absence ( table 1 ) . thus , the immunomodulatory influence of k17 is first manifested at an early stage in this model . topical application of the phorbol ester , 12-o - tetradecanoylphorbol-13-acetate ( tpa)25 , to ear skin induces acute inflammation and epidermal proliferation ( fig . 3a , b ) , providing a tumor - free , dermatitis - like setting in which to assess the impact of k17 loss . the latter curtails hyperplasia - driven epidermal thickening ( wt ear tissue : 34.12.3 m in tpa- vs. 10.40.3 m in vehicle - treated ; k17 ear tissue : 18.7 0.8 m in tpa- vs. 10.60.8 m in vehicle - treated ; fig . markers related to compromised skin barrier function ( s100a826 , thymic stromal lymphoprotein ( tslp)14 , -defensin 27 ) show elevated mrna levels in tpa - treated wildtype skin ( fig . a partial shift toward a th2-dominated cytokine profile is seen in tpa - treated k17 skin , though the magnitude of the changes is less than in gli2 skin . the th1 chemokines cxcl5 , ccl3 and il-1 are reduced 2.4- , 3.0- and 1.7-fold , respectively , and the th2 cytokine il-20 is 7.1 fold higher in tpa - treated k17 skin relative to control ( table 2 ) . skin keratinocytes from gli2 and gli2/k17 newborn mice were seeded for primary culture ( 48h ) , and treated with tpa ( 12h ) to assess whether key changes in cytokine / chemokine expression are keratinocyte - autonomous . under basal conditions , gli2 and tpa induces a two - fold enhancement in gli2 keratinocyte proliferation by 12h , whereas gli2/k17 cells are unchanged ( supplemental fig . levels of cxcl11 , cxcl5 , cxcl9 and cxcl10 mrnas , among others , are significantly lower in tpa - treated gli2/k17 keratinocytes ( fig . these chemokines promote keratinocyte proliferation in skin tumors , and show a tight spatial correlation with k17 expression28,29 . re - expression of k17 into tpa - treated gli2/k17 keratinocytes markedly elevates the levels of cxcl5 , cxcl9 , and cxcl11 mrnas , relative to mock - transfected cells ( fig . k17 impacts the tpa - induced expression of select chemokines relevant to bcc pathogenesis in both adult epidermis in situ and isolated newborn keratinocytes in culture , suggesting that the mechanism(s ) involved are in part cell - autonomous . several nf - kb target genes show a modest but consistent reduction in their expression in gli2/k17 relative to gli2 keratinocytes in tpa - treated cultures ( supplemental figure 5a ) . this is consistent with the prominent role of nf-b in skin inflammatory conditions 30 and , in particular , with its impact on cxcl5 , cxcl9 , and cxcl11 expression3133 . similar analyses of p80 whole ear skin tissue revealed no difference between the genotypes , likely reflecting the large complexity of these lesions in situ and the occurrence of secondary or compensatory changes ( supplemental figure 5 , b , k17 has been shown to promote anagen growth during hair follicle cycling34 and stimulates protein synthesis during tissue repair35 . the phenotype reported here can not be correlated to obvious alterations in these roles , again as inferred from analyses of skin tissue sections ( data not shown ) or extracts ( supplemental fig . 5 , b k17 is ectopically expressed in numerous settings associated with robust inflammation including cutaneous wounds , various carcinomas , psoriasis , and virus - induced warts10 . high levels of k17 expression correlate with a poor prognosis in breast36 and pancreatic37 cancers whether this phenomenon is related to altered inflammatory signatures represents an issue of interest . there exists a correlation between th1 hyperactivity and k17 expression in psoriatic plaques19 ; plaque resolution coincides with a shift to a th2 response and loss of k17 expression . we posit that the presence of k17 in epidermis ( and related epithelia ) promotes hyperplasia in bcc - like tumors ( this study ) and likely in additional tumors and inflammatory disease settings in part through its ability to promote a specific type of inflammatory response . normal contexts in which prominent k17 expression is not correlated to local inflammation ( e.g. , hair follicles ) may benefit from an immune - privileged status38 or reflect its regulation via post - translational modifications or interaction with other proteins34,35 . a role for k17 as an immunomodulator , whether direct or indirect , provides a novel way of conceiving how snps affecting k5 influence the risk of developing bcc 2 , and makes these keratins potentially attractive target for novel therapies aimed at curtailing conditions driven by or linked to chronic inflammation . methods and any associated references are available in the online version of the paper at http://www.nature.com / naturegenetics/.

the sperm protein associated with the nucleus in the x chromosome ( spanx ) gene family encodes for proteins that are not only expressed in germ cells , but also in a number of tumors . in addition , spanx genes map in an interval of the x chromosome ( namely , xq27 ) , which has been found to be associated with familial prostate cancer by linkage analysis . the aim of this study was therefore to evaluate spanx protein expression in normal prostate tissues and in prostate carcinoma . for this purpose , formalin - fixed and paraffin - embedded sections obtained from 15 normal ( at autopsy ) donors and 12 men with prostate cancer were analyzed by immunohistochemistry . about 40% of both normal and tumor prostate samples resulted spanx positive . signals were exclusively within the nucleus in normal prostate cells , whereas both nuclear and cytoplasmic positivity was observed in tumor cells . in conclusion , these findings showed that spanx genes are expressed in both normal and tumor prostate gland , but the latter showed a peculiar cytoplasmic staining positivity . this suggests a possible association between spanx over expression and prostate cancer development . additional studies are needed to corroborate this hypothesis . basaloid skin tumors , including basal cell carcinoma ( bcc ) and basaloid follicular hamartoma ( bfh ) , are associated with aberrant hedgehog ( hh ) signaling1 and , in the case of bcc , an expanding set of genetic variants including keratin 5 ( k5)2 , an intermediate filament - forming protein . we show that genetic ablation of keratin 17 ( k17 ) protein , which is induced in basaloid skin tumors3,4 and co - polymerizes with k5 in vivo5 , delays bfh tumor initiation and growth in mice with constitutive hh signaling in epidermis6,7 . the delay is preceded by reduced inflammation and a polarization of inflammatory cytokines from a th1/th17- to a th2-dominated profile . absence of k17 also attenuates hyperplasia and inflammation in a model of acute dermatitis . re - expression of k17 in gli2tg k17/ keratinocytes induces select th1 chemokines with established roles in bcc . our findings establish a novel immunomodulatory role for k17 in hh - driven basaloid skin tumors that could impact additional tumor settings , psoriasis , and wound repair .

most vertebrates are unable to grow and reach their normal adult form without the thyroid hormone [ 1 , 2 ] . however , various subsequent studies revealed that thyroid hormone plays a key role in rat testis development [ 47 ] . mammalian testis is a target of thyroid hormone action and altered thyroid function which is known to affect testicular functions [ 8 , 9 ] . thyroid hormone is well known as a physiological modulator of oxidative stress [ 10 , 11 ] . previous studies on the role of thyroid hormone in testis are mainly focused on histological and physiological aspects resulting into reproductive failure [ 6 , 8 , 1214 ] . altered thyroid function during early stages of development and maturation may adversely affect testicular growth and physiology [ 79 ] ; particularly the sertoli cells , which play a major role in spermatogenesis and are the main cell type in the testis which expresses t3 receptors . it is well established that the formation of normal numbers of sertoli cells is a key factor in determining testis size , germ cell numbers per testis , and sperm production rate in adulthood in a range of mammals , including humans . in our earlier reports , it was demonstrated that experimentally hypothyroidism modulates several oxidative stress and antioxidant defence parameters in mitochondria and postmitochondrial fractions in adult rat testis [ 18 , 19 ] . we also reported about the effects of transient and persistent hypothyroidism on testicular antioxidant defence system in mature rats to know the role of hypothyroidism - induced oxidative stress in testicular development and maturation . however , information about the role of deprivation of thyroid hormone in early developmental growth and function of testis in relation to antioxidant status in immature rats is inadequate . in rat , spermatogenesis reaches the pachytene stage at 20 days of age . at 25 days of age , round spermatids appear , and at the age of 50 days , mature spermatozoa are released into seminiferous tubular lumen [ 20 , 21 ] . fetal type leydig cells disappear soon after birth ( during first 2 weeks after birth ) and are replaced by adult type leydig cells [ 22 , 23 ] . sertoli cell proliferation reaches its maximum level just before birth and ceases by the age of 3 weeks . hence , four weeks or around 30 days of age , testis is undergoing critical stages of development and thyroid hormone is playing a key role during this stage . in our earlier studies , it was reported that transient hypothyroidism ( from day 1 of neonatal age till day 30 ) modulated testicular antioxidant defence status as well as functions in adult stage . moreover , it was also shown that germ cells in these rats were under oxidative stress and had poor antioxidant defence system . so , it is interesting to know about the testicular antioxidant defence status and oxidative stress parameters along with testicular physiology at the 30 days of age , that is , before puberty . the objective of the present study is to investigate the modulation of antioxidant defence status in neonatal persistent hypothyroid rats before their sexual maturation . in addition , we have tried to identify the specific cell populations in testes vulnerable to degeneration during neonatal hypothyroidism in immature rats . male pups obtained from breeding were made hypothyroid from day 1 of neonatal age till day 30 of postnatal age . hypothyroidism was induced in neonates by feeding the lactating mother with 0.05% 6-n - propyl-2-thiouracil ( ptu ) through the drinking water [ 7 , 18 , 25 ] . from the day of parturition till weaning ( 25 day postpartum ) , the pups received ptu through mother 's milk ( or ) drinking water and then directly from drinking water containing 0.05% ptu for the remaining period of experimentation . adult male wistar rats ( rattus norvegicus ) were divided into two groups containing 15 animals each . group - ii rats were treated with ptu from day 1 postpartum to day 30 postpartum . animal care , maintenance , and experiments were conducted under the supervision of the institutional animal ethics committee ( iaec ) regulated by the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) , government of india . on the 31st day postpartum , body weight of animals in group - i and ii was recorded ; the animals were sacrificed by decapitation , trunk blood was collected and allowed to clot and then centrifuged to obtain sera . the serum levels of total t3 , t4 , tsh , and testosterone were measured by using elisa kits ( monobind , inc . , costa mesa , ca , usa , and equipar diagnostici , italy ) . testes and accessory sex organs , that is , seminal vesicle , ventral prostate and epididymis were removed , cleaned in cold 0.15 mol / l nacl ( normal saline ) , pat dried , and weighed . testes from three animals were pooled to one sample and five such samples were taken for the study . the whole procedure of tissue processing was done as described earlier by sahoo et al . a 20% ( w / v ) homogenate of testis was prepared in 50 mm phosphate buffer , ph 7.4 , containing 0.25 m sucrose . the crude homogenate ( ch ) was centrifuged at 600 g for 10 min to precipitate nuclei and other cellular debris . the resulted supernatant was again centrifuged at 10,000 g for 20 min to separate mitochondria . the mitochondrial pellet was washed thrice in 50 mm phosphate buffer , ph 7.4 ( 10,000 g for 5 min each ) , and finally suspended in the same buffer to obtain mitochondrial fraction ( mf ) . for lipid peroxidation ( lpx ) , lpx was measured in ch and mf by monitoring the formation of thiobarbituric acid - reactive substances ( tbarss ) following ohkawa et al . the assay was performed in presence of 0.02% ( w / v ) butylated hydroxytoluene to suppress artefactual peroxidation during heating . malondialdehyde ( mda ) was used as the standard and tbars were expressed in terms of mda equivalents as nmol tbars formed / mg protein . protein carbonyl ( pc ) content was estimated in testicular ch , mf , and pmf following levine et al . for measuring protein - sh content , pmf and mf samples were first precipitated in ice - cold 5% trichloroacetic acid ( tca ) containing 0.01 m hcl and centrifuged at 1000 g for 15 min . protein precipitates dissolved in 8 m guanidine hydrochloride were then used to measure thiol content and presented as mol / mg protein [ 29 , 30 ] . total glutathione content was measured in the supernatant of tca - precipitated testicular pmf and mf samples by enzymatic recycling procedure and gssg was measured after masking gsh with 2-vinylpyridine [ 31 , 32 ] . total gsh equivalents , gsh and gssg contents were expressed as nmol / g tissue . hydrogen peroxide ( h2o2 ) content was determined in testicular pmf and mf following horseradish peroxidase - dependent oxidation of phenol red . for measuring the activity of catalase ( cat ) , pmf treated with ethanol ( 0.17 m ) and triton x-100 ( 1% ) was directly used . the enzyme activity was measured following decomposition of h2o2 at 240 nm as described earlier and expressed as nkat / mg protein . superoxide dismutase ( sod ) activity in pmf ( for cu / zn - sod ) and mf ( for mn - sod ) of testis was determined following modified nitrite method . one unit of enzyme activity was defined as the amount of enzyme capable of inhibiting 50% of nitrite formation under assay conditions . glutathione peroxidase ( gpx ) activity was assayed in pmf and mf by measuring oxidation rate of nadph in presence of hydroperoxide , gsh , and glutathione reductase ( gr ) [ 19 , 36 ] . total and selenium - dependent gpx activities were estimated by using cumene and tert - butyl hydroperoxides , respectively . the difference between total glutathione peroxidase and selenium - dependent glutathione peroxidase ( se - d - gpx ) activities represents the selenium - independent glutathione peroxidase ( se - i - gpx ) activity [ 19 , 37 ] . glutathione reductase ( gr ) activity in testicular pmf and mf was assayed by measuring oxidation rate of nadph in presence of gssg . in testicular pmf and mf , glutathione - s - transferase ( gst ) activity was measured following change in absorbance of the conjugated product of gsh and 1-chloro , 2 , 4-dinitrobenzene ( cdnb ) at 340 nm . gpx , gr , and gst enzyme activities were expressed as nkat / mg protein . glucose-6-phosphate dehydrogenase ( g6pd ) activity was assayed in pmf by measuring nadph formation from glucose-6-phosphate along with nadp and expressed as nkat / mg protein . following sacrifice , testis tissues were immediately fixed for histological studies in freshly prepared sublimate formal , dehydrated in graded ethanol series , cleared in xylene , and embedded in paraffin wax . all data represent means standard deviation and were subjected to unpaired student 's t - test to find out the level of significance between control and experimental rats . the weight gain in group - ii rats on 31st day of age was almost 50% less than group - i control rats ( table 1 ) . the weight of testis , seminal vesicle , and ventral prostate ( g/100 g body wt ) decreased significantly in ptu - treated rats of group - ii ( table 2 ) . the serum t3 , t4 , and testosterone level decreased significantly whereas the level of tsh increased by several folds in group - ii rats in comparison to control rats in group - i ( table 3 ) . marked decrease in seminiferous tubule diameter was observed in neonatal persistent hypothyroid 30-day - old rats ( group - ii ) as compared to the corresponding controls ( table 4 ) . there was a significant decrease in spermatogonia ( 24% ) and spermatocytes ( 79% ) accompanied by complete absence of round spermatids in group - ii testis ( table 4 ) . however , a significant increase in the sertoli cell number in group - ii rat testis was marked ( table 4 ) . the level of endogenous lipid peroxidation showed a decrease by 13.6% and 64% in crude and mitochondrial fractions , respectively , in response to 30 days persistent ptu treatment ( group - ii ) in comparison to control ( group - i ) rats ( figure 1 ) . a significant elevation in protein carbonyl content was recorded in the crude homogenate ( by 35.6% ) , mitochondrial ( 78% ) , and postmitochondrial fraction ( 22% ) of testis of group ii hypothyroid rats when compared to the controls ( group i , figure 2 ) . hydrogen peroxide content remained unaltered in pmf and decreased ( by 15% ) in mf of group - ii ptu - treated rats ( figure 3 ) . the protein - sh content decreased significantly by 14% and 24% , respectively , in mitochondrial and postmitochondrial fractions of testis of group - ii rats ( figure 4 ) . in mitochondrial fraction of testis of group - ii , rats , total gsh equivalent , oxidized , and reduced glutathione contents and gsh to gssg ratio declined , respectively , by 50% , 16.7% , 67% , and 61% ( table 5 ) . on the other hand , the group - ii testicular postmitochondrial total gsh equivalent and reduced glutathione contents did not alter with 23.5% decrease in gssg activity and 27% elevation in gsh to gssg ratio ( table 5 ) . the mn - sod activity was elevated by 20% in mf whereas cu / zn - sod activity was increased by 30% in pmf of testis of group - ii rats ( figure 5 ) . in the postmitochondrial fraction of testis , the cat activity was increased by 83% in response to ptu treatment in group - ii rats in comparison to control rats ( group - i ) ( figure 6 ) . in group - ii rat however , in postmitochondrial fraction , total gpx and se - i - gpx declined , respectively , by 6.7% and 22.35% with 9.5% elevation in se - d - gpx activity ( figure 7 ) . the gr activity increased significantly by 56% and 55% , respectively , in mitochondrial and postmitochondrial fractions of testis of group - ii rats in response to ptu treatment for 30 days ( figure 8) . the gst activity decreased by 59% in mitochondrial fraction and elevated by 42% in postmitochondrial fraction of testis in response to ptu treatment in group - ii rats when compared to control rats ( group - i , figure 9 ) . an increase in enzyme activity in post mitochondrial fraction of testis was recorded in group - ii rats by 73% ( figure 10 ) . thyroid hormones are reported to play a critical role in growth , differentiation , maturation , and metabolism in vertebrates . essential role of thyroid hormone in male sexual maturation , and reproduction was reported earlier [ 4 , 6 , 42 ] . both hyperthyroidism and hypothyroidism states alter testicular antioxidant defence parameters in adult rats [ 7 , 18 , 19 , 25 , 4345 ] . however , the effects of persistent hypothyroidism on neonatal rats before their puberty need to be studied . the present study reports the effect of neonatal persistent hypothyroidism induced by ptu treatment on the testicular antioxidant system and spermatogenic function in rats before puberty . the efficacy of the treatment was confirmed by a dip in t3 and t4 levels and a consequent increase in tsh level in serum of neonatal persistent hypothyroid rats . ptu is well known to decrease the conversion of peripheral t4 to t3 and thereby reduces serum t3 concentration . a significant reduction in the body weight was observed in ptu - treated rats when compared to control rats as reported earlier in case of neonatal hypothyroidism . the lower h2o2 contents in hypothyroid rats might be due to lower superoxide production in hypothyroid state due to hypometabolic rate . the hypothyroid rat testis resulted in a reduction in lipid peroxidation , which might be as a result of metabolic depression due to hypothyroid condition which serves as a protective factor to prevent lipid peroxidation [ 18 , 47 , 48 ] . in another study , ( 2005 ) also observed a low lipid peroxide content in testicular and renal tissues of hypothyroid rats . on the contrary , oxidative stress is marked as the increase in protein carbonyl contents in crude and mitochondrial fraction of hypothyroid rat testis in the present study . ( 2008 ) , who observed an increased level of carbonylation of protein in crude homogenate of testes of hypothyroid rats . superoxide dismutase ( sod ; ec 1.15.1.1 ) constitutes the first line of coordinated enzymatic defense against ros by dismutating o2 into o2 and h2o2 . catalase ( cat ; ec 1.11.1.6 ) and glutathione peroxidase ( gpx ; ec 1.11.1.9 ) are most crucial for detoxifying h2o2 , thereby preventing the generation of hydroxyl radical by the fenton reaction . selenium - dependent glutathione peroxidases ( se - d gpxs ) are the major selenoprotein - containing gene family in mammals . among the different types of selenium - dependent hydroperoxide - reducing isozymes , phospholipid hydroperoxide glutathione peroxidase ( ph - gpx / gpx-4 ; ec 1.11.1.12 ) and classic cellular glutathione peroxidase ( cgpx / gpx-1 ; ec 1.11.1.9 ) other gpx activities in mammalian systems are selenium independent . the se - independent gpx ( se - i gpx ) , component of gst alfa class ( accession : ipr003080 gst_alpha ) , is responsible for gpx activity in testis . a significant elevation in total sod and cat activities in response to neonatal persistent hypothyroidism and a simultaneous decrease in total gpx , se - d gpx ( gpx-1 and gpx-4 ) , and se - i gpx in the pmf of testis in experimental group suggested that sod and cat have predominant role to fight oxidative stress than gpx in hypothyroid rats . the majority of the cytosolic gpx in rat testis existed as selenium- and nonselenium - dependent gpx which is present in the leydig cells . gpx is primarily responsible for h2o2 removal in testicular mitochondria that does not contain catalase . gpx plays a crucial role in scavenging peroxyl radicals and thereby maintains functional integrity of the cell membrane , spermatogenesis , sperm morphology , and motility . in testis , ph - gpx or gpx-4 which is partially cytosolic and partially bound we observed that mitochondrial se - dependent gpx ( including gpx1 and gpx-4 ) activity was unaltered in hypothyroid rats as reported earlier by chattopadhyay et al . in fact , the role of cgpx / gpx1 in protecting testes from oxidative injury is questionable as cgpx knockout mice were found to be fertile . it has also been suggested that the metabolic pathway of testosterone biosynthesis requires protection against peroxidation and will be affected by a decrease in the gpx activity . the lower serum testosterone level in hypothyroid rats in the present study also corroborates the fact . mammalian testis contains relatively high levels of gsh , which is reported to play an important role in the proliferation and differentiation of spermatogenic cells by protecting them from noxious effects of ros . the level of gsh is different in testicular cell types , having maximum level in sertoli and germ cells . thyroid hormone is known to play a role in triggering the biosynthesis of gsh , therefore under hypothyroid states , it is possible that there may be a fall in gsh levels contributing to susceptibility of the testes to oxidative stress . the ratio of reduced glutathione ( gsh ) to oxidized glutathione ( gssg ) in tissues is also considered as one of the important marker of oxidative stress . the decrease in its ratio and also a decrease in gsh contents in testicular mf of hypothyroid rats suggest severely hindered gsh metabolism . such type of decreased gsh contents in testis - mitochondria of hypothyroid rats was also reported earlier . moreover , declined gsh contents in germ cells were reported earlier in case of transient hypothyroid rats . the decreased protein - sh contents in both mitochondrial and postmitochondrial fractions might be due to the response against disturbed testicular redox pool in hypothyroidism . similar effects on testicular protein - sh during hypothyroidism were reported by chattopadhyay et al . glutathione - s - transferase ( gst ; ec 2.5.1.18 ) metabolizes xenobiotics by conjugating with gsh . in fact , gst - catalysed conjugation of gsh with exogenous compounds and endogenous metabolites such as 4-hydroxynonenal is regarded as major cellular defense mechanism against toxicity . the decreased activity of this enzyme in mf of testes in neonatal persistent hypothyroid rats reflects the inability of the testis to counteract oxidative stress by this pathway . glutathione reductase ( gr ; ec 1.6.4.2 ) is the enzyme responsible for maintenance of gsh by reducing gssg back to gsh . stimulated gr activity in neonatal hypothyroid rats indicated that mitochondrial metabolism attempted reduction of gssg to gsh in order to maintain redox status . such type of gr elevation was also reported in hypothyroid rat testis by sahoo et al . increase in activity of glucose-6-phosphate dehydrogenase ( g6pd ) due to neonatal hypothyroidism might be due to higher demand for nadph as gr requires this reducing equivalence to generate reduced gsh from gssg . the testis seems to be the most vulnerable during neonatal persistent hypothyroidism as evident from the significantly decreased germ cell count . they reported absence of round spermatids in 30 days hypothyroid testis and the inability of the spermatogenic cells to complete meiosis . ( 1991 ) also reported an impaired spermatogenesis and increased degeneration of germ cells in testis in response to prolonged hypothyroidism in rats . increased sertoli cell number in hypothyroid rats in the present investigation might be due to extended sertoli cell proliferation as in an earlier report , van haaster et al . ( 1992 ) and holsberger and cooke ( 2005 ) demonstrated that neonatal hypothyroidism may increase adult sertoli cell populations by extending sertoli cell proliferation . as hypothyroidism causes significant decrease in lh and fsh with a fall in serum testosterone [ 69 , 70 ] , this might lead to a disruption of the spermatogenic and steroidogenic processes . moreover , it has been reported that neonatal hypothyroidism adversely affects leydig cell proliferation , differentiation , along with impaired steroidogenesis [ 71 , 72 ] . hampered of testicular function may have their origins in fetal or early life as a result of abnormal development or proliferation of sertoli cells . a compromised antioxidant defence system marked by increased protein carbonylation , disturbed redox status during neonatal hypothyroidism , might have contributed to poor growth and development of testis by affecting spermatogenesis and steroidogenesis in rats before puberty as indicated by reduced germ cell number , complete absence of round spermatids , decreased seminiferous tubule diameter , and decreased testosterone level . such type of altered testicular physiology by hypothyroidism is reflected in adulthood with hampered fertility as evidenced by reduced total viable germ cells and sperm counts . ewing 's sarcoma family of tumors are a group of small round - cell neoplasms , which include ewing 's sarcoma ( ews ) , primitive neuroectodermal tumor ( pnet ) , askins tumor , pnet of the bone , and extraosseous ewing 's sarcoma ( ess ) . ewing 's sarcoma / peripheral primitive neuroectodermal tumor , presumed to be neuroectodermal in origin , most often occurs in the bone and soft tissues of children and young adults . the intracranial extraosseous ewing 's sarcoma ( cns - ess ) is extremely rare and often misdiagnosed as central nervous system pnet ( c - pnet ) or as other primary intracranial neoplasms . an 11-year - old female presented with a history of headache for one year , which increased in intensity over the last one month . she had repeated episodes of vomiting for one month and a left temporal scalp swelling , which was gradually increasing in size . on examination there was bilateral papilledema , but no other focal neurological deficits were detected . the left temporal scalp swelling was mildly tender , firm , immobile , and non - pulsatile with ill - defined margins . the computed tomography scan ( ct scan ) of the brain showed a rounded , well - defined , heterogeneously hyperdense , enhancing lesion in the left temporoparietal region , with a mass effect and destruction of the left temporal bone , extending into the scalp , suggesting the possibility of a meningioma . no evidence of calcification was noted within the lesion [ figure 1 ] . magnetic resonance imaging ( mri ) of the brain showed a left temporal lesion , hypointense on t1 , heterointense on t2 , with heterogeneous enhancement [ figure 2 ] . a significant mass effect was detected with a midline shift to the right , of 15 mm . preoperative ct scan of the brain showed a rounded , well - defined , heterogeneously hyperdense , enhancing lesion in the left temporoparietal region , with a mass effect and destruction of the left temporal bone extending into the scalp , suggesting the possibility of meningioma . no evidence of calcification was noted within the lesion preoperative mri of the brain showed a left temporal lesion , hypointense on t1 , heterointense on t2 , with heterogenous enhancement the patient underwent left temporoparietal craniotomy and decompression of the lesion . the operative findings suggested that the lesion was extradural , with erosion of bone in some areas and involvement of the overlying temporalis muscle . the histopathological examination showed round - to - oval cells arranged in lobules , separated by a thin vascular channel , having vesicular nuclei , with indistinct nucleoli . it was negative for cd20 , cd3 , myosin , and glial fibrillary acidic protein . the overall features were compatible with a primitive neuroectodermal tumor ( pnet ) , with a possibility of ewing 's sarcoma in the temporal region . the tumor was positive for t ( 11 ; 22 ) ( q24 ; q12 ) translocation detected by fluorescent in situ hybridization ( fish ) in the tumor biopsy sample . the findings of further metastatic workups , including ct scans of the thorax and abdomen and a bone scan with technetium tc-99 m , were negative . ews / pnet 's are characterized by immunoreactivity to the surface antigen cd99 / mic2 , which is expressed in up to 97% of the cases a postoperative ct scan of the brain revealed a large extradural and subdural hematoma in the left frontotemporal region , with a postoperative craniotomy defect and scalp edema . the child went on to receive 12 weeks of chemotherapy with vincristine , doxorubicin , and cyclophosphamide , alternated with ifosfamide and etoposide . the treatment was completed with the same combination of chemotherapeutic drugs for another 24 weeks . james ewing ( 18661943 ) first described the tumor , establishing that the disease was different from lymphoma and other types of cancer known at that time . in 1921 , he described a lethal primary bone lesion that affects children and young adults and most frequently originates in the long bones ( 47% ) , pelvis ( 19% ) or ribs ( 12% ) . the skull is rarely involved , probably in less than 4% of the cases , with the frontal and parietal bones being the most commonly affected . extraosseous ewing 's sarcoma ( ees ) has been recognized as a distinct disease entity that afflicts young adults in the second and third decades of life , with equal sex predilection . the ees commonly involves the paravertebral regions of the spine and in rare instances , these lesions arise in the intracranial compartment , where they have been commonly misdiagnosed as c - pnet , because of the similarity in their histological appearance . very few cases of the central nervous system extraosseous ewing 's sarcoma ( cns - ees ) have been reported in pathology literature.[611 ] jay et al . was probably the first to describe a patient with an isolated posterior fossa mass that histologically resembled a medulloblastoma , but demonstrated the t(11;22 ) ( q24;q12 ) translocation , which confirmed cns - ees . as far as our knowledge goes , this is the seventh case we are reporting . the differential diagnosis of an intracranial round cell tumor is primitive neuroectodermal tumor ( neuroblastoma ) , lymphoma , rhabdomyosarcoma , and ewing 's sarcoma . the histological examination reveals that these tumors are composed of small , undifferentiated neuroectodermal cells and frequently demonstrate immunohistochemical and/or electron microscopic features of glial or neuronal differentiation . recent advances in the molecular classification has allowed a clear pathological distinction between c - pnet and cns - ees . cns - ees is known to demonstrate in 97% of the patients , a strong membrane expression of the mic-2 gene product , designated cd99 , which is specifically recognized by the monoclonal antibodies o13 and hba71.[1316 ] in addition , the chromosomal translocation t(11 , 22)(q24;q12 ) , detected by fish , is found in more than 90% of ees . this nonrandom translocation is not found in the central primitive neuroectodermal tumors ( c - pnet ) such as the medulloblastoma and supratentorial pnet . although , cns - ees is histologically similar to c - pnets such as the medulloblastoma , it differs significantly in clinical behavior , treatment , and prognosis . the treatment options available for patients with cns - ees are similar to those of ees elsewhere in the body and include multimodality treatment comprising of surgery , chemotherapy , and radiation . patients with c - pnet also require surgery ; however , the chemotherapeutic and radiation therapy protocols differ from those used for cns - ees . multiagent chemotherapy regimens including cyclophosphamide , ifosfamide , doxorubicin , dactinomycin , and etoposide have been shown to be effective in localized ewing 's sarcoma , in various trials . the primary treatment for localized ess consists of neoadjuvant chemotherapy , with a combination of vincristine , doxorubicin , and cyclophosphamide , alternating with ifosfamide and etoposide , given for 12 to 24 weeks , followed by definitive local treatment of surgery or radiation , or surgery and postoperative radiation directed at the primary site . the treatment is completed with administering chemotherapy with a similar combination for a total of 36 to 49 weeks . because of the small number of patients , the prognosis of cns - ees is not clearly known , although it has been suggested that patients with ees that arises from structures within or around the cns may have a more favorable outcome than patients with c - pnet .

altered thyroid function during early stages of development is known to affect adversely testicular growth , physiology , and antioxidant defence status at adulthood . the objective of the present study is to investigate the modulation of antioxidant defence status in neonatal persistent hypothyroid rats before their sexual maturation and also to identify the specific testicular cell populations vulnerable to degeneration during neonatal hypothyroidism in immature rats . hypothyroidism was induced in neonates by feeding the lactating mother with 0.05% 6-n - propyl-2-thiouracil ( ptu ) through the drinking water . from the day of parturition till weaning ( 25 day postpartum ) , the pups received ptu through mother 's milk ( or ) drinking water and then directly from drinking water containing ptu for the remaining period of experimentation . on the 31st day postpartum , the animals were sacrificed for the study . an altered antioxidant defence system marked by elevated sod , cat , and gr activities , with decreased gpx and gst activities were observed along with increased protein carbonylation , disturbed redox status in hypothyroid immature rat testis . this compromised testicular antioxidant status might have contributed to poor growth and development by affecting the spermatogenesis and steroidogenesis in rats before puberty as indicated by reduced germ cell number , complete absence of round spermatids , decreased seminiferous tubule diameter , and decreased testosterone level . ewing 's sarcoma / peripheral primitive neuroectodermal tumors occur most often in bone and soft tissues of children and young adults . the intracranial manifestation of the disease is rare , and when present , this is often misdiagnosed with other varieties of primary brain tumors . we report such a case of extraosseous ewing 's sarcoma , which was initially suspected to be a case of meningioma in an 11-year - old girl .

nitric oxide ( no ) is known to have several important vasculo - protective effects ( el - mas et al 1997 ; grdal et al 2005 ) . reduced production of no may lead to atherosclerosis ( lscher and vanhoutte 1990 ; harrison 1997 ; shimokawa 1999 ) . the synthesis of no from the amino acid , l - arginine , is catalysed by no synthase ( nos ) . no then plays a critical role in the relaxation of vascular smooth muscle cells ( vsmcs ) . it is well known that no is involved in reducing vsmc proliferation , adhesion of platelets and leukocytes ( moncada and higgs 1993 ; draijer et al 1995 ; myers and tanner 1998 ; brocq et al 2008 ) . no is likely , therefore , to be an important contributory factor in the pathogenesis of some forms of stroke . there are 3 distinct isoforms of nos identified in humans : endothelial ( enos ) , neuronal nos ( nnos ) and inducible nos ( inos ) . it synthesizes very low levels no under basal conditions ( lscher and vanhoutte 1990 ; harrison 1997 ; shimokawa 1999 ) . because enos plays a central role in the maintenance of vascular homeostasis , including regulation of the cerebral circulation , a number of studies have been conducted to investigate the genetic association between the enos gene and the incidence of ischemic stroke . howard and colleagues ( 2005 ) detected 3 single nucleotide polymorphisms ( snps ) in the promoter region , 1 in exon 7 ( g894 t ) and 1 insertion / deletion polymorphism within intron 4 among 110 cases with ischemic stroke and 206 controls . they found strong associations of both the 922 g - a and 786 t - c snps with ischemic stroke . hou and colleagues ( 2001 ) were the first to report the genetic association between the enos gene and ischemic stroke in a chinese population and a recent work has confirmed the intial finding ( berger et al 2007 ) although the enos association failed to be replicated in a japanese population ( yahashi et al 1998 ) . this study might have been hampered by poor replication , a common problem with genetic analysis . linkage disequilibrium ( ld ) structure and sample power may be one of major reasons for poor replication of an initial finding . therefore , the present study was designed to apply a large sample size to investigate genetic association between the enos gene and ischemic stroke . the present study recruited 560 unrelated patients with ischemic stroke and 153 unrelated controls in order to undertake a genetic analysis for association between the nos3 gene and ischemic stroke . patients with ischemic stroke were admitted to the first hospital of jilin university , changchun , china in the period between 2005 and 2006 . they were diagnosed as having ischemic stroke independently by at least two well - trained neurologists based on strict neurological examination , computed tomography , or magnetic resonance imaging , which meet the international classification of diseases ( nchs 2007 ) . detailed information regarding their medical history , medication and clinical presentation was taken through either their close relatives or themselves . of these 560 patients ( 398 males and 162 females ) , 426 were suffering from simple lacunar infarction , 246 from large - vessel infarction and 112 from both . the study was granted prior approval by the ethics committee of jilin university , changchun , china . to perform genetic analysis , we selected snp rs3918181 ( rsa i site ) as a genetic marker at the nos3 locus . it is an a to g base change present in intron 14 of the gene . polymerase chain reaction ( pcr)-based restriction fragment length polymorphism ( rflp ) analysis was performed to genotype rs3918181 . genomic dna used for pcr amplification was extracted from the whole blood sample using a dna extraction kit ( promega , beijing , china ) . pcr amplification was performed in a 25-l reaction volume containing 10 mm tris - hcl ( ph 8.3 ) , 50 mm kcl , 1.5 mm mgcl2 , 0.001% ( w / v ) gelatin , 200 m of each dntp , 0.4 m of each primer , 1.0 unit of taq dna polymerase ( promega , beijing , china ) , and 3050 ng of genomic dna . the conditions used for pcr amplification included an initial denaturation at 94 c for 5 min , followed by 3540 cycles of 94 c for 45s , 5560 c for 1 min and 72 c for 1 min , and a final elongation at 72 c for 10 min . a 15-l aliquot of the pcr products was completely digested with 68 units of restriction enzymes . the hardy - weinberg equilibrium for the genotypic distributions of rs3918181 was tested using the chi - square ( ) goodness - of - fit test . applied to test sample power for individual alleles was tested using the spss samplepower 2.0 software package ( spss inc . , allelic association was tested by the cocaphase module of the unphased program ( dudbridge 2003 ) and genotypic association was tested by the spss program for windows 15.0 ( spss inc . ) . the goodness - of - fit test showed that the genotypic distributions of rs3918181 were not deviated from hardy - weinberg equilibrium in both the patient group ( = 0.166 , p = 0.684 ) and the control group ( = 0.421 , p = 0.517 ) . these 560 case and 153 control samples gave a power of 60% for detection of small effect size and 100% for detection of medium effect size . analysis with the cocaphase program showed that rs3918181 was associated with ischemic stroke in males ( = 4.04 , p = 0.044 , or = 1.43 , 95%ci 1.012.02 ) and frequency of allele a of rs3918181 was significantly higher in male patients than male control subjects ( table 2 ) . however , female patients did not show such an association ( table 2 ) . as shown in table 3 , the test revealed genotypic association between rs3918181 and ischemic stroke in males ( x = 4.26 , df = 1 , p = 0.039 , or = 1.61 , 95% ci 1.022.53 ) but not the genotypic association was not observed in female subjects . the gene coding for enos is mapped to the long arm of chromosome 7 ( 7q36 ) and spans 21 kb of dna , although it contains 26 exons . it should be sufficient to capture a linkage disequilibrium ( ld ) signal by genotyping a highly polymorphic dna marker present in the middle of the gene . snp rs3918181 is located in intron 14 of the gene and has heterozygosity of 0.43 . this finding gives further evidence in support of the enos association with ischemic stroke in the chinese population , although female patients did not show such an association in our samples ( tables 2 and 3 ) . the sample power test indicates that the study is not sufficiently powered at present for detection of small effect - size gene . however , we will continue to collect controls matched for age , sex and ethnicity in order to confirm the results and 80% of power will be needed for combined samples in our future study . it is located at the interface of these two vastly different environments , and thus is charged with making the final decision about what , from an uncontrolled and often hostile chemical surround , should be incorporated into the highly controlled biochemical environment within . taste is the beginning of a long chemosensory tube that extends from palate to intestines , with receptors along that length that are sensitive to the products liberated by digestion . those on the palate are not unique to taste ; the same receptors often occur elsewhere in the body . what is unique is that those serving taste gather their information before the irrevocable decision to swallow has been made , and so can influence that decision . hence , whereas the distribution of information from other chemical sensors may be limited to the gastrointestinal ( gi ) tract , or may be conveyed through the vagus only to the hindbrain , that from taste receptors is projected through the brainstem to the thalamus and multiple cortical sites as well as to ventral forebrain areas . this vast distribution through the central nervous system permits the control of somatic and autonomic reflexes , a cognitive evaluation , and hedonic appreciation . an apt metaphor for taste is a janus head , mounted on an ancient city gate , one face turned outward to assess the traffic beyond the walls , warning of approaching toxins and alerting gatekeepers to the availability of nutrients ; the other turned inward to monitor the city 's changing needs and to adjust its decisions of what passes through the city walls to satisfy them ( scott , 1987 ) . this view of the role of gustation as a visceral ( internal ) as well as somatic ( external ) sense defines its learning capacity . taste is exquisitely well suited to learn from visceral consequences ( satiety , nausea ) ; it is less inclined to learn from those that are somatic ( tones , lights , and shocks ) . as garcia noted in describing the development of a taste aversion following a meal , no other aspect of the event was implicated in having caused nausea : not his dinner companions , the table settings , or the background music , only the visceral component represented by the taste of the meal ( garcia et al . , 1985 ) . the realm of conditioning can be broadly divided into those events that one can do something about , and those that one can not . the former typically demands operant conditioning to manipulate the environment to one 's satisfaction , using somatic senses to gather information and striated muscles for action ; the latter more commonly demands classical conditioning to prepare for the inevitable , often using smooth muscles . gustatory learning serves as a special case of classical conditioning , with taste representing the conditioned stimulus ( cs ) , and the visceral sequelae of ingestion , the unconditioned stimulus ( us ) . it has been proposed that the visceral response alters the reward value of the taste , and that this new value then guides the animal 's behavior to either seek or avoid that taste ( rolls , 2005 ) . such learning can occur in an appetitive or aversive direction , with the establishment of either conditioned taste aversions ( ctas ) or preferences ( ctps ) . it is of greater urgency to the animal to avoid a chemical that has sickened it than to develop a preference for one among many that have proven to provide nutrition . the cta is readily established with a single pairing of the gustatory cs and the visceral us , even with a cs - us interval for several hours . it is not impaired by placing the animal under deep anesthesia or rendering it comatose before the us is delivered . indeed , the predisposition of an animal to develop a cta is so striking that the investigators first suspected it to be an artifact , a suspicion laid to rest only by an exhaustive series of studies and arguments ( revusky , 1977 ) . as easily as it is created , a cta is notoriously difficult to extinguish ( nolan et al . , 1997 ) . the cta also has a robust impact on behavior , often suppressing subsequent acceptance of the cs to less than 10% of preconditioned levels , even in animals motivated to consume by moderate deprivation . with such a dramatic impact on behavior , the cta has a half - century history as a rich topic of research . thousands of studies were conducted during the 1960s and 1970s on behavioral variables such as the distinctiveness and novelty of the taste , the nature and severity of the nausea , the amount of time between them and how that time was spent . with these clearly defined , the cta could be employed by researchers as a tool for altering taste acceptability , creating a profound reduction in acceptance of the cs , from which generalization gradients of both quality and intensity could be determined to reveal the relative similarities among taste qualities . in parallel , behavioral neuroscientists began to investigate the mechanisms by which this extraordinary learning process occurred , using rats in nearly all studies . they performed lesions of taste pathways and relays to determine which areas of the nervous system were required in order to develop and retain a cta . there followed electrophysiological investigations of the impact of a cta on taste processing , immunohistochemical studies of gene expression elicited by a cta , and microdialysis experiments on the neurochemical consequences of the experience . a ctp can be established rather quickly by pairing a novel taste with recovery from a dietary deficiency , most notably the provision of thiamine to animals on a thiamine - deficient diet ( rodgers , 1967 ; capretta , 1977 ) . more commonly , however , the impact of a ctp on behavior is revealed only gradually over days of continuous pairing of taste with nutrition , though that impact can reach levels equal to those of a cta in the opposite direction , i.e. , approaching 100% preference ( sclafani and nissenbaum , 1988 ) . the electrophysiological and neurochemical concomitants of a ctp are also more subtle than those of a cta . yet , the ctp may have played a larger role in defining human culture than the cta , for while the latter is powerful , it is idiosyncratic to the individual . the ctp , by contrast , is often shared by members of a culture where certain foods are available . it is typical of a culture 's cuisine that there are a few piquant tastes ( the cs ) accompanied by carbohydrate loads ( the us ) . the gustatory cs comes to be favored by association with the nutritional us , and the cuisine , with all its cultural trappings and traditions , tends to bind its consumers together as part of their cultural identity . in the paragraphs that follow , i will review some of the work on the mechanisms of ctas and ctps that have come from our laboratory and those of our colleagues . the ingredients of the cta taste and visceral distress are represented widely across the cns . investigators have performed lesions of areas that receive such inputs in an effort to define which are crucial to the creation and to subsequent retention of a cta . results implicate the area postrema in acquisition ( rabin et al . , 1983a ) , but not retention ( rabin et al . , 1983b ) they reveal that loss of the parabrachial nuclei ( pbn ) causes the most profound disruption on both creating a cta ( with lesions of the lateral division ) ( spector et al . , 1992 ; yamamoto et al . , 1994 ) and maintaining a previous aversion ( medial division ) ( sakai et al . , 1994 ) . they implicate the ventromedial globus pallidus in both acquisition and retention ( hernadi et al . , 1997 ) . electrolytic lesions of the basolateral amygdala disrupt the creation and retention of a cta ( yamamoto et al . , 1995 ) , but nmda lesions , which spare fibers of passage , do not ( chambers , 1990 ) ; thus the amygdala remains a likely participant in cta formation , but the axons that pass through it may be of greater import , since leaving them intact sustains the learning . lesions of prefrontal cortex have yielded conflicting results , and its role in ctas remains uncertain . those in insular cortex ( ic ) , however , reliably degrade cta acquisition ( braun et al . , 1982 ; bermudez - rattoni and mcgaugh , 1991 ) and have an even larger impact on retention . thus , the cast of participants in creating and retaining a cta as demonstrated by these fixed , permanent lesions range from the deepest recesses of the brain stem through ventral forebrain to the neocortex . greater insight may be gleaned from lesions that are reversible , or that combine the loss of more than one area . ivanova and bures ( 1990a , b ) temporarily disabled regions of the brainstem with microinjections of tetrodotoxin ( ttx ) . they reported that ttx injected in the pbn up to one day in advance or four days following training blocked the consolidation of a cta without affecting the rejection threshold for quinine . thus , taste processing per se remained intact , but the associative functions required for learning the aversion were disrupted by inactivation of the pbn . the crucial role of pbn in mediating associative taste learning was reinforced by clark and bernstein ( 2009 ) . these investigators performed asymmetrical , unilateral lesions of the pbn and ic , and thereby disrupted cta acquisition . however , when both lesions were made in the same hemisphere , leaving the contralateral hemisphere intact , learning proceeded normally . the effect of a cta is to reverse the behavioral reaction to a previously preferred taste to one of the revulsion . the rejection response is organized in caudal brainstem ( norgren and grill , 1982 ) and released in stereotypical fashion upon encountering an inherently aversive stimulus or one to which a cta has been formed . the nucleus of the solitary tract ( nst ) , the first central relay for taste , is likely to be involved in cta formation . here gustatory and visceral afferents converge ( norgren , 1981 ) yet do not directly overlap , communicating instead via the adjacent reticular formation , offering a close association between signals from the two necessary components of a cta . chang and scott ( 1984 ) took single neuron recordings from the nst of rats that were ( 1 ) unconditioned ( tasted the saccharin cs with no subsequent nausea ) , ( 2 ) pseudoconditioned ( experienced the nausea us with no preceding taste ) , or ( 3 ) conditioned ( the taste of the saccharin cs was paired with licl - induced nausea us ) . the recordings revealed that sweet - oriented nst neurons gave exaggerated responses to the saccharin cs in conditioned rats , and that the increase was due to a sharp spike of activity that peaked at about 900 msec following stimulus delivery ( figure 1 ) . moreover , the neural response profile to saccharin in conditioned rats was more similar to those of aversive stimuli . we concluded that the sensory code for the saccharin cs ( and , to a lesser extent , for other sweet stimuli ) was altered at the first central taste relay by conditioning , and that such a modification could explain not only the behavioral reaction , but also the immunohistochemical , and neurochemical consequences of a cta described below . such a modification of the taste signal might also reveal why the cephalic phase insulin release from the pancreas , a parasympathetic reflex elicited by sweet taste , is blocked after a cta has been created to that taste ( louis - sylvestre and lemagnen , 1980 ) . the altered neural message would lose the capacity to innervate the vagal efferents responsible for stimulating pancreatic cells . post - stimulus time histograms of the mean responses among sweet - sensitive neurons in the nucleus of the solitary tract to the conditioned stimulus : 0.0025 m sodium saccharin . activity is shown for control ( dotted line ) , pseudoconditioned ( dashed line ) , and conditioned ( solid line ) rats . the enhanced activity during the first three seconds of the evoked response in conditioned animals may represent the increased salience of the sodium saccharin taste and its aversive quality . in a subsequent experiment , we created , and then fully extinguished a cta to saccharin ( nolan et al . , 1997 ) , then recorded responses to saccharin and other stimuli in these recovered rats and in unconditioned controls ( mccaughey et al . , 1997 ) . the mean responses to all stimuli were no different in the two groups of rats , nor was there any significant difference between the neural response profiles to any taste . however , the neural activity was not completely restored to the preconditioning state . there remained a clear vestige of the conditioning experience in an attenuated burst given to the cs by the sweet - sensitive subgroup of neurons . the burst was no longer associated with conditioned behavior which was fully extinguished though it may have served as a permanent marker for a once - salient cs that can abet subsequent reacquisition of the aversion . curiously , lesions in gustatory nst do not interfere with either the acquisition or retention of a cta ( grigson et al . , 1997 ; shimura et al . , 1997a ) . even if the electrophysiological effects recounted above were only advisory to a more commanding cta nucleus or circuit , the blockade of taste information at this obligatory synapse would appear to prevent any subsequent learning . the lack of an impact may reflect the inadequacy of lesions to fully compromise a functioning relay , particularly when they must spare adjacent areas that control vital reflexes , including respiration . in the parabrachial nucleus , the creation of a cta to nacl resulted in an elevated response to that stimulus in sodium - specific taste cells , in agreement with the responses to the saccharin cs in the nst described above ( shimura et al . , 1997b ) . the same was found in the ic ( yamamoto et al . , 1989 ) and amygdala , where an exaggerated response to the cs is often expressed as inhibition ( yasoshima et al . , 1995 ) . finally , in the hypothalamus of nave rats , the taste of saccharin activates areas associated with feeding and inhibits those for satiety ; after the saccharin is paired with nausea , these roles are reversed ( aleksanyan et al . , 1976 ) . thus , the impact of a cta is demonstrable in the electrophysiological activity of neurons across the widely dispersed regions that process taste activity , and that impact is appropriate to guide the aversive reaction to the cs that follows conditioning . the formation of long - term memories requires the expression of immediate early genes and the synthesis of their associated proteins ( mcgaugh , 2000 ) . the gene c - fos has been shown to be expressed and the associated fos protein synthesized in a variety of species as a basis for modifying the neural activity associated with learning ( sanyal et al . , 2002 ) . c - fos expression , then , can serve as a useful index of conditioning . 1994 ) demonstrated that sucrose elicited c - fos expression in the nst after it had been paired with an intraperitoneal injection of licl . this was not simply a response to the aversive taste that sucrose had become , for quinine did not induce the same expression in unconditioned rats ( houpt et al . , 1996 ) . however , this index of learning in nst was blocked when the amygdala was impaired , demonstrating the importance of centrifugal fibers in mediating the conditioning process ( schafe and bernstein , 1996 ) . his colleagues have shown that c - fos is induced in cells in the medial division of pbn by hedonically positive tastes associated with ingestion , and in neurons in more lateral divisions by aversive tastes . saccharin activates medial cells in nave rats , but after the saccharin has been paired with a licl injection , its taste induced c - fos expression in the lateral division ( yamamoto et al . , 1994 ) . moreover , when c - fos expression was blocked in pbn , cta learning was impaired , just as it is when the pbn is lesioned ( yasoshima et al . , 2006 ) . thus , a functioning pbn , capable of modifying its responses to a taste stimulus as a consequence of experience , is crucial to learning to avoid toxins . in forebrain , bernstein and koh ( 2007 ) addressed the issue of which areas reacted to novel tastes differently from those that were familiar , since only the former serve as effective conditioned stimuli . they used c - fos expression to identify the central nucleus of the amygdala ( cna ) and ic as two such sites . the next question was which of these also responded to the us , the second of the necessary elements of a cta . only the cna expressed c - fos to a licl us , implicating this nucleus as a crucial nexus for making the association . yet while ic , which had responded to gustatory novelty , did not show increased c - fos expression to the us alone , its neurons did respond to pairing of the cs and us , reinforcing its role in the associative process , perhaps through communication with the pbn and amygdala ( ferreira et al . , 2006 ) . gene expression , of course , is a precursor of protein production , and it is assumed that the protein is the basis for forming the associative memory . accordingly , when protein synthesis was inhibited by administration of anisomycin into the ic , a cta was not formed ( rosenblum et al . , 1993 ) . more specifically , the administration of oligodeoxynucleotide ( odn ) antisense to c - fos in the amygdala blocked cta acquisition ( lamprecht and dudai , 1996 ) . again , the two critical forebrain regions ic and amygdala are implicated , and the capacity of their neurons to synthesize proteins in general , and fos in particular , is essential to associative learning . finally , bernstein and her colleagues ( barot et al . , 2008 ; used a direct visualization approach to identify neurons that responded both to the cs and us , reasoning that this afforded them prima facie data from which to support the association . they found such cells only in the basolateral nucleus of the amygdala , and only under normal training conditions , i.e. , cs followed by us . the bulk of evidence from gene expression studies , then , focuses on three structures as being central to the acquisition and retention of ctas : the pbn , the amygdala ( both central and basolateral nuclei ) , and the ic . it is unsurprising , then , that when the cs to which a cta has been developed is subsequently presented , reminiscent of that experience , plasma corticosteroid levels rise ( smotherman et al . , 1976 ) . yet , the cta does not depend on this adrenal index of stress , for adrenalectomized rats acquired ctas as readily as controls ( ader et al . , 1978 ) . a more specific measure of the impact of a cta is seen in the levels of neurochemicals associated with reward or aversion , particularly in the limbic system . reward is associated with increased dopamine ( hoebel , 1984 ) and reduced acetylcholine ( rada et al . , 1991 ) in the nucleus accumbens , and lowered serotonin levels in the paraventricular hypothalamus ( stanley et al . , 1989 ) . this relationship has been demonstrated both through microdialysis , where dopamine levels in the accumbens rose following a sweet taste , and conversely through reverse microdialysis in which dopamine administered to the accumbens increased sucrose consumption ( hajnal and norgren , 2001 ) . the taste of saccharin evoked dopamine release in the rat 's accumbens , in accord with its reinforcing value . however , if the saccharin had been paired with nausea to create a cta , the same stimulus caused a reduction in dopamine , and instead a release of acetylcholine in accumbens ( mark et al . , 1991 ) , and serotonin in the hypothalamus ( west et al . , 1991 ) , denying the neurochemical basis for reward . when subjects develop a cta , there is little doubt that they are subsequently repulsed by the taste . not only do they avoid it , they also show the well - defined mimetic reflexes of aversiveness : gaping , head - shaking , and chin - rubbing . all that follows the blockade of parasympathetic reflexes , the alteration of the afferent signal and its projection to brain areas associated with avoidance , the reversal of neurochemical release from rewarding to aversive is in accord with this powerful experience . the behavior changes only over days of training , though the cs does finally reach asymptotically high levels of acceptance , and is quite resistant to extinction . mimetic responses of rats , however , do not change as the cs wins acceptance , raising the question of whether the hedonic quality of the cs has increased ( sclafani , 1991 ) . ( 1997 ) recorded electrophysiological responses from the nst of rats that had been conditioned to prefer either of two formerly aversive chemicals . in each case , the gustatory code was modified to reflect a less aversive quality , though the effect was less pronounced than that seen in the opposite direction upon creation of a cta ( chang and scott , 1984 ) . secondly , the taste for which a preference was acquired now elicited a heightened dopamine release in the nucleus accumbens , a clear neurochemical marker of reward ( mark et al . , 1994 ) . the animal knows two facts : what the chemical was ( taste ) , and what it did ( gi ) . this information permits it to tailor its chemical selection to full individual advantage over a lifetime . the learning process draws on responses that extend from the viscera through caudal brainstem , to ventral forebrain and cortex , implying an ancient system , much like the control of feeding itself . it is only a marginally conscious process , for ctas can be learned while comatose , and most people can not recall the occasion upon which they developed a food aversion ( bernstein , 1985 ) . conditioned aversions and preferences provide the operative link between the chemical and biochemical environments . 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 .

we recruited 560 unrelated patients with ischemic stroke and 153 unrelated controls to undertake a genetic analysis for association between the nos3 gene and ischemic stroke . all the subjects were chinese of han descent . because the nos3 gene spans about 21 kb of dna and contains 26 exons , we selected a single nucleotide polymorphism ( snp ) rs3918181 , an a to g base change located in intron 14 of the gene , as a dna marker . pcr - based restriction fragment length polymorphism analysis was applied to genotype rs3918181 ( rsai site ) . the chi - square ( 2 ) goodness - of - fit test showed that the genotypic distributions of the marker were not deviated from hardy - weinberg equilibrium in both the patient group ( 2 = 0.166 , p = 0.684 ) and the control group ( 2 = 0.421 , p = 0.517 ) . the cocaphase analysis showed allelic association of rs3918181 with ischemic stroke in males ( 2 = 4.04 , p = 0.044 , or = 1.43 , 95% ci 1.012.02 ) and frequency of allele a was significantly higher in male patients than male control subjects . the 2 test revealed genotypic association between rs3918181 and ischemic stroke in males ( 2 = 4.26 , df = 1 , p = 0.039 , or = 1.61 , 95% ci 1.022.53 ) but not in females . the present work suggests that rs3918181 is associated with ischemic stroke in male patients . this finding gives further evidence in support of the enos association with ischemic stroke in the chinese population . taste is the final arbiter of which chemicals from the environment will be admitted to the body . the action of swallowing a substance leads to a physiological consequence of which the taste system should be informed . accordingly , taste neurons in the central nervous system are closely allied with those that receive input from the viscera so as to monitor the impact of a recently ingested substance . there is behavioral , anatomical , electrophysiological , gene expression , and neurochemical evidence that the consequences of ingestion influence subsequent food selection through development of either a conditioned taste aversion ( cta ) ( if illness ensues ) or a conditioned taste preference ( ctp ) ( if nutrition ) . this ongoing communication between taste and the viscera permits the animal to tailor its taste system to its individual needs over a lifetime .

a 36-year - old man visited the emergency department of inje university haeundae paik hospital with severe pain in both lower limbs for 1 day . a physical examination revealed pale , pulseless , and cold lower limbs . one year ago , he had been diagnosed with kimura disease ( kd ) after excision of a right upper limb mass ( fig . the initial laboratory findings were as follows : leukocyte count of 45.510/l with 74% eosinophils , elevated serum fibrinogen degradation product ( fdp ) level ( 18.7 g / ml ) , elevated d - dimer level ( 5.1 g / ml ) , and extremely elevated serum immunoglobulin ( ig ) e ( > 2,500 iu / dl ) . a computed tomography ( ct ) angiogram showed total occlusion of both popliteal arteries ( pas ) ( fig . under the presumptive diagnosis of acute limb ischemia ( ali ) , the patient underwent an emergency thrombectomy of both femoral arteries using a fogarty catheter ( edwards laboratories , santa ana , ca , usa ) . after the operation , the pain and color changes resolved , but the right foot drop remained . to prevent thromboembolic events , immunochemical tests for autoimmune diseases , including lupus anticoagulant , anticardiolipin igg and igm antibodies , anti - beta2-glycoprotein igg and igm , and anti - phospholipid antibodies , were negative . protein s activity was low , but the levels of the total and free protein s antigen were normal , suggesting a type - ii protein s deficiency . an echocardiogram was performed ; it revealed no evidence of a cardiac thrombus or decreased systolic function of the left ventricle ( ejection fraction=47% ) . under the presumptive diagnosis of kd - related ali , the patient was administered steroid therapy . a follow - up ct angiogram on postoperative day ( pod ) brachial index ( abi ) was 0.87 on the right side and 0.62 on the left . coronary angiography ( cag ) on pod 5 showed triple - vessel disease with chronic total occlusion of the left anterior descending artery ( lad ) , left circumflex artery ( lcx ) , proximal obtuse marginal artery , and posterior descending artery ( fig . stent insertion for the coronary artery was delayed because we needed to observe the kd activity and plan the second operation for the left pa . we performed the second operation on pod 17 because of the low abi of the left lower limb . a thrombus and a hypertrophic , injured intimal layer were observed after the longitudinal arteriotomy ( fig . after the removal of the thrombus and the injured intimal layer , a patch angioplasty using bovine pericardium ( vascu - guard biovascular inc . , st . 2e ) : ( 1 ) thrombosis occluding the vessel lumen and intimal thickening with fibroblastic proliferation ( the intima showed reactive changes with fibroblastic proliferation ) were observed . ( 2 ) the thrombotic material contained a prominent number of eosinophils , and mixed inflammatory cell infiltration composed of eosinophils and lymphocytes was observed in the vessel wall . two months after the first discharge , the patient returned to the emergency department with right lower extremity pain and a color change that had developed the day before . a ct angiogram showed total occlusion from the right superficial femoral artery to the distal pa ( fig . we performed an emergency thrombectomy , partial endarterectomy , and patch angioplasty on the right side . the operative findings on the right side were the same as the findings on the left side . a coronary stent was inserted into the lad , and the lcx was balloon dilated ( fig . the patient was then treated with aspirin , clopidogrel , and cilostazol for preventing in - stent thrombosis and prednisolone for kd . kd is characterized by marked eosinophilia , an elevated ige level , and recurrent subcutaneous nodules around the head and neck . however , the increased serum ige level and eosinophilia suggest an allergic disease [ 14 ] . despite an unclear mechanism , hypereosinophilia can lead to a hypercoagulable condition and vasculitis according to some published reports [ 57 ] . a possible mechanism is that the cytokines secreted by eosinophils , including eosinophil cationic protein , eosinophil peroxidase , and major basic protein , activate platelet aggregation . some researchers have demonstrated that human eosinophils contain various amounts of the tissue factor ( tf ) and have concluded that relatively high tf expression in patients with hypereosinophilia may contribute to an increased thrombotic risk . in this case however , kd has been treated with local mass excision , steroids for systemic inflammation , revascularization , and anticoagulation for organ ischemia . some researchers have reported that radiotherapy was a successful treatment for kd . in our patient , furthermore , we performed a partial endarterectomy and patch angioplasty for revascularization . additionally , the patient was administered prednisolone to control the activity of kd . his serum leukocyte and eosinophil counts and fdp and serum ige levels normalized . many revascularization methods are available for ali , including thrombectomy , stent insertion , patch angioplasty , and extra - anatomic bypass with a synthetic or saphenous vein graft . patch angioplasty is more effective for removing a thrombus and widening the lumen than a stent and is easier than a bypass . cag to evaluate other forms of vasculitis revealed coronary artery obstructive disease ( caod ) , and we inserted a coronary stent instead of performing a bypass . because of the patient s young age , coronary artery bypass grafting was the treatment of choice for caod . the mechanism of caod in our patient was different from that of caod in other cases . however , antiplatelet therapy was needed , and the patient required long - term follow - up . in conclusion , kd is a systemic disease that affects many organs and presents with thromboembolism and vasculitis . in a patient with kd , physicians should evaluate the vascular system , including the coronary arteries . businesses offering goods and services to consumers often request feedback from former customers to assess strengths and weaknesses and to leverage positive feedback and testimonials as marketing materials . as these testimonials become more digitized and more public on sites like yelp , facebook , and google+ , businesses have a high stake in ensuring that their consumers are satisfied with the quality of service or goods they provide [ 2 , 3 ] . in the healthcare space , the hospital consumer assessment of healthcare providers and systems ( hcahps ) initiative provides a survey tool to measure patients ' perspectives and satisfaction within hospital systems . the hcahps survey aims to produce standardized data on patients ' perspectives on hospital care to allow for meaningful comparisons between hospitals based on consumer importance and publicly represent these findings to encourage transparency and incentivize hospital systems to improve their quality of care . the hcahps initiative has identified 21 rating items that have been determined to affect patient perspectives on hospital care and has generated a 32-question survey that assesses quality of care from nurses and doctors , hospital environment , and experiences in the hospital . while this survey instrument is widely used in the hospital setting , an equivalent tool does not exist to evaluate satisfaction and perspectives in other areas of medicine and health , including addiction rehabilitation programs . unique in its function , residential addiction treatment centers simultaneously operate as healthcare providers and hospitality managers , delivering medical and psychological support while offering accommodations , meals , and amenities in lieu of a clients ' typical home environment . in this exploratory study , we aim to determine which criteria are most significant to influencing overall alumni satisfaction with an inpatient drug or alcohol rehabilitation facility . because the residential and intensive outpatient addiction rehabilitation industry functions at the crux of social science , medicine , and hospitality services , we hypothesize that alumni perceptions of facility amenities and perceived quality of individual therapies and group counseling will have significant influence on alumni satisfaction . we also hypothesize that the presence of subsequent relapse after discharge from a facility as a proxy measurement for success will negatively influence alumni satisfaction . a survey was created using surveymonkey 's online survey design platform to assess alumni feedback and satisfaction of a recent stay in an inpatient drug or alcohol rehabilitation facility . the survey contains 45 questions with an equal mix of single choice and open - ended questions . the survey also includes three matrix questions for evaluation of facility features , individual therapy , and group counseling . respondents were asked to evaluate their facilities by the following criteria : the facility 's amenities and individual and group counseling services . they were also asked to respond to demographic and personal questions regarding age of entry into treatment , method of payment , and motivation to enter treatment . the evaluated aspects are as follows : amenities accommodations , meals and nutrition , exercise options , leisure / extracurricular , holistic offerings ( e.g. , yoga and meditation ) , staff support , connectivity ( internet / phone use ) , visitor policy , and cleanliness;individual counseling counselor availability , counselor training / experience , counselor respect for patient 's treatment preferences , flexibility to switch counselors , inclusion of holistic approaches , and inclusion of alternative / creative treatment approaches;group counseling quality of lead counselor , frequency of meetings , consistency of meetings , member empowerment / safety , and conflict resolution . amenities accommodations , meals and nutrition , exercise options , leisure / extracurricular , holistic offerings ( e.g. , yoga and meditation ) , staff support , connectivity ( internet / phone use ) , visitor policy , and cleanliness ; individual counseling counselor availability , counselor training / experience , counselor respect for patient 's treatment preferences , flexibility to switch counselors , inclusion of holistic approaches , and inclusion of alternative / creative treatment approaches ; group counseling quality of lead counselor , frequency of meetings , consistency of meetings , member empowerment / safety , and conflict resolution . the survey targets alumni of inpatient drug or alcohol rehabilitation facilities whose most recent enrollment occurred within the past ten years . respondents were targeted through surveymonkey audience , which is a service that provides survey takers with targeted , representative sample populations . additionally , respondents were asked a qualifying question , to ensure they met targeting requirements . inclusion criteria for analyses include a minimum survey completion of 75% over the variables forming the aggregate scores ( i.e. , category components ) and 100% over the remaining relevant variables . survey data collected from august 25 , 2014 , to february 22 , 2015 , were included in these analyses . of the 864 survey responses received , 485 responses did not fit the sample parameters for completeness and were removed from the database , resulting in a sample of 379 surveys . recoding was required to convert the survey platform 's default coding into a consistent format compatible with stata 's requirements . full details regarding cleaning and coding ( .log ) are available at the reader 's request . data were analyzed using a variety of logistic regression models , due to the outcome variables being dichotomous . for the purpose of these analyses , the dataset was declared as survey data , using stata 's svyset command . all of the following analyses utilized the appropriate svy command prefix , unless otherwise noted . the likelihood of a respondent responding positively ( i.e. , answering yes ) to the binary questions would you recommend the facility to a friend or family member ? was assessed by examining the impact of the respondent 's perception of the facility 's offerings . to better evaluate this potential impact , variable was formed by finding the mean of the respective category component scores for each respondent . all data were deidentified , and respondents provided consent for use of survey data in public reporting and research directly on the survey . two metrics were considered indicators of satisfaction with the facility attended : if treatment was worth the cost , and if the respondent would recommend this facility to a friend or family member who is seeking addiction treatment . these two metrics were considered indicators of satisfaction as recommendations and personal cost - benefit ratios are standard proxies for satisfaction in marketing practices , as well as in health service assessments [ 4 , 814 ] . 66.05% ( n = 249 ) of respondents entered inpatient addiction treatment between 40 and 59 years of age . treatment was sought by individuals most frequently at their own behest ( 62.33% n = 235 ) and was most commonly paid for through private ( 33.86% n = 128 ) or government - provided ( 18.25% n = 69 ) insurance . the most frequently occurring profile is an individual that entered treatment as a personal choice between the ages of 40 and 59 years . the second most frequently occurring profile is an individual that entered treatment as a personal choice , between the ages of 26 and 39 years ; this profile fits 13.87% ( n = 52 ) of respondents . likert - scale scores were calculated for each individual metric regarding amenities , individual counseling , and group counseling ( table 2 ) . amenities received an overall composite likert - scale score of 3.99 ( sd = 0.97 ) . respondents scored four out of nine amenities factors highly ( likert - scale score > 4.00 ) . with regard to individual counseling offerings , which received an overall score of 3.84 ( sd = 1.14 ) , respondents highly rated counselor training and experience ( 4.36 sd = 1.10 ) , as well as their respect for the individual 's treatment preferences ( 4.25 sd = 1.18 ) . respondents were less pleased with counselors ' willingness to incorporate holistic approaches ( 3.18 sd = 1.62 ) . group counseling offerings were given an overall score of 4.37 ( sd = 1.00 ) . each of the five factors of group counseling was given a score greater than 4.00 , with the frequency of group meetings receiving the highest likert - scale score . four models were constructed using logistic regression to evaluate factors impacting an individual 's notion of treatment being worth the cost . the first model regresses the three summed aggregate offering scores regarding amenities ( variable name = afacilityscore ) , individual counseling ( acounsscore ) , and group counseling ( agrpcounsscore ) on the variable for was treatment worth the cost ? these variables are did the facility deliver the promised amount of counseling ? ( promcouns ) ; did psychiatric care meet expectations ? ( psyexpect ) ; and did marketing materials accurately portray the facility 's offerings ? ( market ) . the fourth model adjusts model 1c , taking into account whether or not the respondent had relapsed . the results of each model are as follows:(i)model 1a the aggregate mean group score is statistically significant , with a p value of 0.022 . the group counseling score produced the highest degree of substantive significance ; each point increase of the group counseling score corresponds with an increase in the odds of the respondent regarding their treatment as worth the cost by 126%.(ii)model 1b all three variables are statistically significant . whether or not the facility provided the promised amount of counseling has the highest degree of substantive significance . moving from no to yes on this variable corresponds to approximately a 37-fold increase in the odds of the respondent regarding his treatment as worth the cost.(iii)model 1c combining the previous two models yields interesting results . notably , when adjusting for offerings scores , the marketing materials accuracy becomes statistically insignificant . additionally , psychiatric care is no longer significant to the 0.001 level ; it also loses degrees of substantive significance . when taken together , this suggests that these variables are not the strongest predictive components . promised amount of counseling and aggregate mean group counseling score remain statistically significant and increase in substantive significance.(iv)model 1d model suggests that relapse is not a predictive component of whether or not the respondent considers their treatment as worth the cost , as relapse is not statistically significant . furthermore , adjusting for relapse increases the substantive significance of aggregate mean group counseling score and psychiatric expectations score . reproducing this model without the svy - estimation and conducting a likelihood ratio test with 1c yield a statistically insignificant result , suggesting that the model is not an improvement over 1c . model 1a the aggregate mean group score is statistically significant , with a p value of 0.022 . the group counseling score produced the highest degree of substantive significance ; each point increase of the group counseling score corresponds with an increase in the odds of the respondent regarding their treatment as worth the cost by 126% . model 1b all three variables are statistically significant . whether or not the facility provided the promised amount of counseling has the highest degree of substantive significance . moving from no to yes on this variable corresponds to approximately a 37-fold increase in the odds of the respondent regarding his treatment as worth the cost . notably , when adjusting for offerings scores , the marketing materials accuracy becomes statistically insignificant . additionally , psychiatric care is no longer significant to the 0.001 level ; it also loses degrees of substantive significance . when taken together , this suggests that these variables are not the strongest predictive components . promised amount of counseling and aggregate mean group counseling score remain statistically significant and increase in substantive significance . model 1d model suggests that relapse is not a predictive component of whether or not the respondent considers their treatment as worth the cost , as relapse is not statistically significant . furthermore , adjusting for relapse increases the substantive significance of aggregate mean group counseling score and psychiatric expectations score . reproducing this model without the svy - estimation and conducting a likelihood ratio test with 1c yield a statistically insignificant result , suggesting that the model is not an improvement over 1c . four models were constructed using logistic regression to determine potentially influential factors on alumni 's willingness to recommend the facility where they received treatment , represented by the variable recff . the first model regresses the aforementioned three aggregate mean offering scores ( afacilityscore , acounsscore , and agrpcounsscore ) on recff . the second model regresses the three aforementioned expectation variables ( promcouns , psyexpect , and market ) on recff . the results of each model are as follows : model 2a each point increase of the group counseling score corresponds with an increase in the odds of the respondent recommending the facility by 256% . the other two variables are not statistically significant.model 2b all three included variables are significant to , at least , the 0.001 level . of these variables , the most influential is the variable representing the promised amount of counseling ; a respondent answering yes to this question corresponds to an increase in the odds that they would recommend the facility approximately 661%.model 2c by increasing model specification , a clearer picture of recff 's predictors becomes apparent . for the group offering score and promised amount of counseling , the relative consistency in odds ratios and statistical significance across the two models suggests that these variables are influential factors in a facility receiving a positive alumni recommendation . this model also suggests that the respondent 's perceptions of the facility 's amenity offerings and individual counseling are not predictor variables , as these variables produced a statistically insignificant output over both models . finally , when controlling for the offerings scores , the psychiatric expectation and accuracy of marketing materials variables are not statistically significant , suggesting that they may not be strong predictor variables , despite being statistically significant predictors in model 2b.model 2drelapse is not a significant predictor . adjusting for relapse has minimal substantive effects on the significant predictor variables . as with 1d , reproducing this model without the svy - estimation and conducting a likelihood ratio test with 2c yield a statistically insignificant result , suggesting the model is not an improvement over 2c . model 2a each point increase of the group counseling score corresponds with an increase in the odds of the respondent recommending the facility by 256% . model 2b all three included variables are significant to , at least , the 0.001 level . of these variables , the most influential is the variable representing the promised amount of counseling ; a respondent answering yes to this question corresponds to an increase in the odds that they would recommend the facility approximately 661% . model 2c by increasing model specification , a clearer picture of recff 's predictors becomes apparent . for the group offering score and promised amount of counseling , the relative consistency in odds ratios and statistical significance across the two models suggests that these variables are influential factors in a facility receiving a positive alumni recommendation . this model also suggests that the respondent 's perceptions of the facility 's amenity offerings and individual counseling are not predictor variables , as these variables produced a statistically insignificant output over both models . finally , when controlling for the offerings scores , the psychiatric expectation and accuracy of marketing materials variables are not statistically significant , suggesting that they may not be strong predictor variables , despite being statistically significant predictors in model 2b . adjusting for relapse has minimal substantive effects on the significant predictor variables . as with 1d , reproducing this model without the svy - estimation and conducting a likelihood ratio test with 2c yield a statistically insignificant result , suggesting the model is not an improvement over 2c . the respondents ' evaluations of their respective facility 's offerings shed insight into the population 's perception of inpatient treatment . the study results indicate trends in facility offerings and respondents ' perceptions . in the aggregate , this finding is consistent with reviews across other industries , where the literature has shown that the general reviews tend to have a negative skew distribution . alumni reception towards facilities ' amenities and individual counseling is generally positive , albeit to a less enthusiastic degree compared to group counseling . facilities ' individual counseling offerings were the least enthusiastically received , particularly in regard to the facilities ' inclusion of holistic , alternative , and creative approaches . this dissatisfaction with holistic offerings is also present in the amenities category , indicating that such an attitude is pervasive and present in the respondents ' overall perception of the facility . models 1a d aim to determine the most substantial predictors of satisfaction with the financial value provided by treatment . these models suggest that high - quality group counseling , as well as meeting or exceeding expectations for the amount of counseling received , is the most substantial and substantively significant predictors of satisfaction relating to cost worthiness . additional statistically significant predictors of satisfaction for this metric are psychiatric care meeting expectations and , to a lesser extent , individual counseling offerings . models 2a d suggest key factors in alumni 's willingness to recommend a facility to their friends or family members . specifically , the results suggest that the respondent 's perceptions of their group counseling experiences , whether or not the facility delivered its promised amount of counseling and , to a slightly lesser extent , whether psychiatric care meets expectations , are instrumental in alumni 's decision to recommend their facility . the selected outcome variables and their responses are proxy measures of consumer satisfaction with treatment facilities ' services . evaluating the models related to these two variables simultaneously highlights strong and consistent potential predictors of consumer satisfaction with treatment facilities ' services . for both outcomes , and across each relevant model , the aggregate group counseling score and whether the facility kept its promises regarding individual counseling are statistically significant predictors of a respondent 's ( i.e. , consumer 's ) satisfaction with his or her treatment . this consistency across outcomes and models suggests the presence of predictive power for these two aspects of treatment services . to a lesser extent , this consistency is also observed for variables representing individual counseling and whether the facility delivered the expected psychiatric care . these results are strengthened by the failure to confirm the second hypothesis ; adjusted for the other selected variables , whether the respondent had relapsed or not was not a statistically significant predictor of alumni satisfaction . furthermore , its inclusion with these other potential predictor variables may not serve as necessary or beneficial specification . facilities should not approach their clients ' satisfaction with fatalistic uncertainty that the client 's postdischarge actions will solely create a negative retrospective perception of treatment ; rather , facilities should recognize that their actions can directly and primarily impact satisfaction , regardless of long - term client outcomes . it is well documented in the literature that higher patient satisfaction scores correlate well with medical treatment adherence [ 16 , 17 ] . for those struggling with addiction , facilities should aim to provide the most satisfactory service to promote treatment adherence . while individual counseling and amenity offerings certainly should not be ignored , high - quality group counseling should be supported and promoted to enhance clients ' treatment experience . improving satisfaction with group counseling offerings has the highest likelihood of improving overall consumer satisfaction , which in turn is likely to support greater adherence to a recovery plan a goal that should guide facilities ' operations . through incorporating empirical research into operational policy the importance placed on group counseling offerings should not solely be viewed through the lens of predicting consumer satisfaction , as broader implications for the treatment space exist . in addition to the aggregate group counseling score being the most consistent substantively and statistically significant predictor , each of its components , collectively , had the highest mean perception scores of the three aggregate categories . viewed together , these findings suggest that consumers are most enthusiastic and receptive to group counseling offerings . investigations into the role of social support on treatment adherence within the addiction space , and across several other health and wellness industries , have demonstrated moderate to significant improvements in adherence [ 1821 ] . just as satisfaction can improve adherence , adherence and subsequently resulting treatment successcan positively influence retrospective perceived satisfaction , thus beginning a cyclical pattern for satisfaction and treatment adherence [ 22 , 23 ] . for treatment providers , the construction of social support during treatment may build the foundation for successful and satisfactory long - term recovery . future research should aim to determine how group therapies improve satisfaction scores and treatment adherence to encourage clients ' recovery . this study includes the limitation that only two metrics were chosen as proxies for satisfaction . while cost worthiness and recommendations are significant proxies for satisfaction , other proxies may also exist . subsequent investigations may include the use of other metrics indicating satisfaction to test our findings . respondent recruitment utilized a self - selection method , which could contribute to selection bias within the study 's sample population . specifically , as the survey was hosted online , respondent demographics will reflect general internet user demographics , which may not be reflective of the general population . precise demographics for both the general population of rehabilitation alumni are unavailable , limiting the ability to determine sample representativeness . additionally , as reviews tend to skew positive , the potential predictor factors discussed below may not be generalizable for unenthusiastic alumni . through producing multiple models in regard to two outcome variables , our results highlight potentially key predictors of alumni 's satisfaction with their treatment experience . across both outcome variables , alumni 's perceptions of the facility 's group offerings and the facility keeping its promises regarding individual counseling are statistically and substantively significant . the alumni 's perceptions of the facility 's marketing materials ' accuracy and whether the facility delivered the expected psychiatric care are also statistically and substantively significant , but inconsistently so . satisfaction with treatment may be correlated with increased treatment adherence and subsequent long - term success in recovery . the addiction treatment space would benefit greatly from further research into the impact of these predictors , through refining of the aggregate component measurements , as well as evaluating the impact of components themselves , to gain a more clear understanding of predictors of consumer satisfaction and thus a better understanding of the treatment space .

kimura disease ( kd ) is an immune - mediated chronic inflammatory disease of unknown etiology . kd has many complications associated with hypereosinophilia , including various forms of allergic reactions and eosinophilic lung disease . additionally , hypereosinophilia is associated with hypercoagulability , which may lead to thromboembolic events . a 36-year - old man with kd presented with acute limb ischemia and coronary artery occlusion . he underwent thrombectomy , partial endarterectomy of both popliteal arteries , and coronary artery stent insertion . kd is a systemic disease that affects many organs and presents with thromboembolism and vasculitis . in a patient with kd , physicians should evaluate the vascular system , including the coronary arteries . objective . the primary objective of this investigation is to determine which individual and aggregate factors of residential addiction treatment centers are most significant influencers of alumni satisfaction . design . survey targeted alumni of residential addiction treatment facilities . alumni were queried through a survey , which utilized likert - scale matrices and binary response options : 379 respondents met the completion threshold . alumni rated amenities and individual and group counseling factors ; additionally , respondents provided feedback on two satisfaction proxies : cost worthiness and future recommendations . descriptive and relational analyses were conducted , with the latter utilizing logistic regression models . results . individual factors ' scores of group counseling , and overall aggregate group counseling score , are most enthusiastically positive . group counseling is also the most significant influencer of satisfaction . other significant influencers of satisfaction are met expectations for individual counseling and psychiatric care offerings . conclusions . while individual counseling and facility amenities should not be ignored , group counseling may be the most significant influencer of alumni satisfaction . long - term outcomes are not single - faceted ; however , treatment providers should be encouraged to invest in high - quality group counseling offerings in order to best satisfy , and thereby empower , clients .

the majority of japanese patients with type 2 diabetes mellitus are not obese , as reported by kosaka and ogata more than 50 years ago . eastern asian subjects might share common characteristics of the disease , comparable to those reported in korean people . it is , therefore , debatable whether we can apply epidemiological evidence obtained from studies of caucasian subjects with type 2 diabetes and obesity to eastern asian people , especially in the fields of diabetic complications , which are influenced not only by hyperglycemia but also by obesity . for example , type 2 diabetes is a relative risk factor for cardiovascular disease in asians , as in western societies , but the absolute risk differs greatly between these populations [ 47 ] . recently , the legacy effect of intensive glycemic control early after the diagnosis of diabetes was advocated based on the ukpds follow - up study . however , there is no report about the legacy effect of past obesity over a lifetime on diabetic complications . although there have been inconsistent results as to whether obesity is a risk for diabetic nephropathy [ 912 ] , it was reported that current obesity and maximum past body mass index ( bmi ) were significant risk factors for diabetic nephropathy in the japanese . although caucasian subjects with type 2 diabetes usually maintain their body weight status during their disease course , the majority of patients of eastern asian ethnicity begin to lose body weight from around the time of diagnosis of diabetes [ 3 , 13 ] . this was easily overlooked , even if the effect of obesity in the past persisted for a long time , because they were already nonobese at the start of clinical follow - up . to clarify the influence of past obesity on diabetic complications is an important clinical concern in patients of eastern asian ethnicity . we therefore analyzed the difference in the prevalence of diabetic nephropathy in japanese type 2 diabetics according to their history of body weight status to clarify the effect of past obesity on diabetic nephropathy . we examined subjects with type 2 diabetes whose estimated glomerular filtration rate ( egfr ) was 30 ml / min/1.73 m or more and who were admitted to saiseikai central hospital from january 1999 to december 2004 ( n = 1834 ) or keio university hospital from april 1998 to september 2010 ( n = 1093 ) for the management of metabolic control . the study protocol was reviewed and approved by the ethics committee of both hospitals . those subjects in whom the etiology of renal disease was strongly suspected to be other than diabetic nephropathy were excluded . according to the history of body weight status and the japanese criteria for obesity , we defined current obesity as bmi on hospitalization of 25 or more , previous obesity as bmi on hospitalization of less than 25 and self - reported maximum bmi in the past of 25 or more , and continuously lean as maximum bmi of less than 25 . hba1c level on admission was determined by high - performance liquid chromatography ( hplc : arkray inc . , kyoto , japan ) according to the recommended method by the japan diabetes society ( jds ) at that time and converted to the national glycohemoglobin standardization program ( ngsp ) value . egfr ( ml / min/1.73 m ) was calculated as 194 cr age ( with further multiplication by 0.739 for female subjects ) using the equation provided by the japanese society of nephrology . subjects with albumin excretion rate ( aer ) of 20 g / min or more in 24-hour urine were considered to have diabetic nephropathy . hypertension was defined as systolic blood pressure > 140 mmhg , diastolic blood pressure > 90 mmhg , or the prescription of antihypertensive medication . < 1.04 mmol / l , or the prescription of lipid - lowering medication . continuous variables are expressed as mean sd . differences in baseline characteristics among the obesity categories were analyzed by anova and chi - squared test . chi - squared test was also performed to evaluate differences in prevalence , with bonferroni 's correction for post hoc multiple comparisons . as a result , logistic regression analysis with forced entry method was performed to detect significant independent predictors of diabetic nephropathy . we adopted as covariates factors such as sex , age , disease duration , hypertension , dyslipidemia , hba1c , and obesity status ( current obesity , past obesity , and continuously lean ) in this study . we analyzed a total of 2927 persons ( males 2038 , females 889 , age 59.3 10.6 years , bmi 24.0 4.0 , duration of diabetes 10.4 28.0 years , hba1c 9.3 2.8% ) . the prevalence of current obesity in the total subjects was 33.6% , previous obesity 41.5% , and a continuous lean state 24.8% . the prevalence of nephropathy was significantly different among the categories , and both currently obese ( 40.6% ) and previously obese ( 35.6% ) patients had a significantly higher prevalence of diabetic nephropathy than that in continuously lean patients ( 24.3% ) ( p < 0.017 ) . when we divided the patients into quartiles according to current bmi , the prevalence of nephropathy significantly increased as current bmi increased ( figure 1 , p < 0.001 ) . when we similarly divided them into quartiles according to previous maximum bmi , the prevalence of nephropathy significantly increased as previous maximum bmi increased ( figure 1 , p < 0.001 ) . current bmi and previous maximum bmi were highly correlated with each other ( r = 0.785 , p < 0.001 ) . in logistic regression analysis , both current obesity and previous obesity revealed a significant odds ratio for nephropathy , as well as diabetic retinopathy , independent of sex , age , disease duration , hypertension , dyslipidemia , and hba1c ( table 2 ) . we confirmed that previous obesity , as well as present obesity , was closely associated with nephropathy in type 2 diabetes . the notable finding of this study was that obesity is an independent risk factor , not only if it is present , but also if it was present in the past . the mechanism is the theme for investigation of how obesity in the past can influence diabetic complications over time . both current bmi and previous maximum bmi were associated with the nephropathy , as demonstrated in figure 1 . however , so , generally , the higher the previous bmi , the higher the current bmi . when we analyzed the effects of previous obesity , we had to separate the effects of current obesity from those of previous obesity . this was the reason we analyzed the effect of previous obesity according to the categories defined as previous maximum bmi and current bmi . as a result , we could elucidate the effect of previous obesity on diabetic nephropathy . whether nephropathy in type 2 diabetes really derives from diabetes has always been a point of discussion . however , type 2 diabetes per se is a disease so closely linked with obesity and the metabolic syndrome that we can not strictly distinguish the cause among the components of the syndrome . we here found that the effect of obesity was independent of the existence of diabetic retinopathy , and we expect this result can be applied to all type 2 diabetes patients . several groups , including us , have reported that albuminuria was the strongest predictor of the progression of diabetic nephropathy [ 1822 ] . we did not chronologically follow the decline of glomerular filtration rate ( gfr ) in this study . however , as we defined diabetic nephropathy by albuminuria , obesity , either current or past , might relate to the gfr decline through albuminuria . although this concept is strictly defined with exclusion of both nephrosclerosis and diabetic nephropathy , the suspected mechanisms , including the contraction of efferent glomerular arterioles by the activated renin - angiotensin system ( ras ) and glomerular hyperfiltration , as well as glomerular hypertrophy due to insulin resistance , are very similar to those of diabetic nephropathy . the border between the concepts of diabetic nephropathy and obesity - related nephropathy is unclear , and they can not be distinguished clinically if a patient has both . vivante et al . reported that overweight state and obesity in adolescents were associated with significantly increased risk for both diabetic and nondiabetic esrd during a 25-year period . if obesity even before the diagnosis of diabetes influences kidney function later , these concepts are continuous and indivisible . there are some limitations of this study . one is that this study was retrospective , based on self - reported body weight in the past . as for the other limitation , we might have to consider how they lost their body weight because the study subjects required metabolic interventions for poor glycemic status . however , we only have the data of maximum body weight and the body weight on admission but not the duration . so the hypothesis needs to be confirmed in a future large cohort study with more detailed information . in spite of these limitations , we therefore believe it includes important suggestions on the effect of obesity on diabetic nephropathy . our study indicated that obesity in the past , as well as present obesity , was a risk factor for diabetic nephropathy . we should consider the effect of earlier obesity on diabetic nephropathy even if it has been present before the diagnosis of diabetes . monoamine oxidases ( mao ) are mitochondrial enzymes responsible for the metabolic breakdown of monoamines ( serotonin , norepinephrine , and dopamine ) in neuronal tissues causing depletion of monoamines and clinical depression ; while mao inhibitors ( maois ) result in the clinical improvement of mood states . interestingly , drugs that possess maoi properties but are used for purposes unrelated to antidepressant activity are furazolidone ( an antiinfective ) ; procarbazine ( drug used for hodgkin 's disease ) ; and linezolid ( an antibiotic used for serious infections ) . we report a case of serious adverse drug interactions between escitalopram and linezolid precipitating near - fatal serotonin syndrome ( ss ) in an elderly female . relevant literature was retrieved via pubmed , embase , and psycinfo using the keywords ; linezolid , escitalopram , serotonin syndrome supplemented with a manual search of cross references . a 65-year - old elderly female , from urban and middle socioeconomic background , presented with history suggestive of depressed mood , middle and late insomnia , anorexia , lethargy , reduced interest in pleasurable activities since 1 year . she was diagnosed as depressive disorder as per icd-10 diagnostic criteria . she had no prior medical history of hypertension , diabetes , or any drug intake . escitalopram ( 5 mg / day ) and clonazepam ( 0.25 mg / day ) therapy showed partial improvement , fortnight later . escitalopram was increased to 10 mg / day , while clonazepam was discontinued due to drowsiness , a month later . patient had full remission of depressive symptoms with escitalopram ( 10 mg / day ) , 2 months later . patient presented with acute onset high - grade fever , cough with greenish - yellow expectoration , breathlessness , and asthenia of a week 's duration following recent travel . she had received antipyretics , mucolytics , expectorants , antibiotics ( amoxicillin and clavulanate ) for 5 days from medical practitioner but found no respite . later , she was referred to physician and hospitalized . on admission , she had 101f temperature , pulse rate of 126/min , and blood pressure ( bp ) of 136/88 mm hg . her general physical and systemic examination revealed no other abnormal findings , except for bilateral scattered fine crepitations and reduced bilateral air entry in lower respiratory areas . her blood glucose ( 105 mg / dl ) , blood urea ( 25 mg / dl ) , and serum creatinine ( 1.0 mg / dl ) were within normal limits . patient was treated with intravenous linezolid ( 600 mg twice daily ) , antipyretics , bronchodilators ( etophylline ) , expectorants ( guaiphenesin ) , mucolytics ( ambroxol ) , and chest physiotherapy . within the first 24 hours of antibiotic treatment , the patient had a rapid clinical deterioration with restlessness , diaphoresis , tremor , shivering , myoclonus , diarrhoea , exaggerated deep tendon reflexes , hypoxia ( spo2 - 80% with 6 - 8 l of oxygen ) and high fever ( 103f ) , along with mental status changes such as disorientation , confusion , and hallucinatory behavior . however , in view of neurological symptoms , cranial computerized tomography and lumbar puncture for the exclusion of central nervous system ( cns ) infection were performed but were unremarkable . the patient was intubated due to severe respiratory difficulties and transferred to the intensive care unit . detailed history and sequence of emergence of events ( including history of depression and escitalopram treatment from caregivers ) were observed and ss was suspected . linezolid and escitalopram were discontinued , and cyproheptadine ( 4 mg thrice daily ) via the nasogastric tube was initiated . pneumonitis was treated with intravenous cephalosporin ( cefotaxime ; 2 g twice daily ) and aminoglycoside ( amikacin ; 500 mg twice daily ) antibiotics . patient gradually regained consciousness and orientation to person , location , and time . at discharge , vital parameters were stable with pulse of 86 bpm and bp of 130/80 mm hg . in our case , addition of antibiotic linezolid ( 600 mg twice daily ) for the treatment of acute onset pneumonia in a patient receiving serotonergic antidepressant , escitalopram ( 10 mg / day ) for depressive disorder since 2 months , temporally led to the constellation of the neurological and mental state features in the absence of other cns pathology led to the diagnosis of ss . we ruled out the possibility of other drug interactions , as none of them ( paracetamol , etophylline , guaiphenesin , ambroxol ) possess either adrenergic or serotonergic properties supplemented by literature search . linezolid is a totally synthetic compound that was initially synthesized as a reversible maoi class antidepressant . due to its weak , nonspecific mao inhibition , concomitant therapy with an adrenergic or serotonergic agent or consuming tyramine ( > 100 mg / day ) may increase the risk of ss . it is believed to act through early inhibition of protein synthesis via binding to the 23s portion of the 50s ribosomal bacterial rrna subunit inducing conformational structural changes and preventing trna to enter and functionally bind to the ribosome therefore inhibiting mrna translation . its oral bioavailability is nearly equal to intravenous administration , with plasma half - life of 4 - 6 h. peak serum concentrations attain about 20 mcg / ml on 600 mg twice daily dosing in adults . linezolid is metabolized via oxidation procedure in a way independent of cytochrome p450 ( cyp-450 ) ; consequently there is no possible pharmacokinetic mechanism of interaction between linezolid and other medication metabolized through cyp450 pathways . although 80% of the dose of linezolid appears in the urine , dose modification has not been currently recommended in renal insufficiency as serum concentrations and half - life of the drug are not appreciably affected . however , linezolid and its breakdown products are eliminated by dialysis ; the drug should be administered after hemodialysis . linezolid is generally well - tolerated , with some minor side - effects like gastrointestinal disturbances , headache , and rashes . rarely , myelosuppression including anaemia , leukopenia , thrombocytopenia , and peripheral as well as optic neuropathy have been reported on prolonged use ( > 8 weeks ) . escitalopram is the s - enantiomer of racemic citalopram with antidepressant activity through selective serotonin reuptake inhibition ( ssri ) , a drug category that is believed to act through boosting of serotonin neurotransmission via blockade of serotonin reuptake pump , therefore theoretically more prone to be involved in the development of ss . it is 56% plasma protein bound with plasma half - life of 27 - 32 h and reaches steady state plasma levels in 7 days . the primary cyp isoenzymes involved in its metabolism are cyp 3a4 , cyp 2d6 , and cyp 2c19 . escitalopram displays linear and dose - proportional pharmacokinetics for single and multiple doses in the dose range of 10 - 30 mg / day . its clearance in elderly subjects ( > 65 years ) in single - dose and multiple - doses showed approximate increase of 50% in half - life . ss is a disorder typically caused by the combination of two or more medications with serotonergic properties due to increased serotonin release . it is characterized by restlessness , myoclonus , hyperreflexia , diaphoresis , shivering , tremor , and mental status changes , such as confusion . it usually consists of a constellation of neurological and mental state symptoms and commonly diagnosed according to the widely accepted criteria , as summarized in table 1 . the pathophysiological mechanism of ss does not include idiosyncratic , neither idiopathic nor pharmacokinetic drug reactions , but is considered to be a predictable and preventable pharmacodynamic consequence of the excess of serotonergic agonism in cns and peripheral serotonergic receptors . symptoms usually improve with the withdrawal of the predisposing drug agents plus supportive care , as there is no specific evidence - based treatment of the ss . cyproheptadine is a h1 histamine receptor antagonist as well as a nonspecific serotonin receptor antagonist , may have a role in the management of ss ( 4 mg thrice daily ) . widely accepted diagnostic criteria for serotonin syndrome concomitant use of maois ( including linezolid , furazolidone , and procarbazine ) along with adrenergic / serotonergic antidepressants , st . john 's wort , sibutramine , or opiate drugs that have ssri - like properties ( meperidine , tramadol , methadone , dextromethorphan , propoxyphene , and fentanyl ) have been reported to precipitate dangerous ss . rarely , ss may also be precipitated by concomitant use of ssris with metoclopramide , sibutramine , fenfluramine or dexfenfluramine , lithium , dihydroergotamine , sumatriptan ; but risk is severe when ssris and maois are combined . linezolid has been reported to induce ss as an interaction with almost every category of antidepressant medications like imipramine , amitriptyline , fluoxetine , citalopram , escitalopram , paroxetine , sertraline , l - tryptophan ( precursor of serotonin ) , mirtazapine , trazodone , buspirone , venlafaxine , and duloxetine . psychiatrists need to be aware that all ssris ( except fluoxetine ) should not be co - administered with maois within 2 weeks of discontinuing either drug , lest may precipitate ss . fluoxetine due to its long half - life is recommended to be stopped at least 5 weeks prior to initiation of maois and conversely , maois are to be discontinued at least 3 weeks prior to initiation of fluoxetine . however , in patients receiving ssris who acutely require linezolid therapy for short - term ( 10 - 14 days ) therapy , coadministration with careful monitoring is reasonable because ssris generally require tapering to avoid discontinuation symptoms . physicians utilizing these drugs ( linezolid , furazolidone , procarbazine ) need to be sensitized about potentially fatal ss when coadministered with antidepressant therapy . clinicians need to exercise reasonable degree of care and skills to prevent such interactions primarily and also be competent to detect early and manage such adverse events , lest may amount to medical negligence .

aims . we analyzed the prevalence of nephropathy according to past body weight status in japanese subjects with type 2 diabetes because the influence of past obesity on diabetic complications is not certain . methods . we examined the prevalence of nephropathy in 2927 subjects with type 2 diabetes mellitus according to current bmi and maximum bmi in the past . we defined current obesity as bmi on hospitalization of 25 or more , previous obesity as bmi on hospitalization of less than 25 and self - reported maximum bmi in the past of 25 or more , and continuously lean as maximum bmi of less than 25 . results . the prevalence of nephropathy was significantly higher in subjects with current obesity ( 40.6% ) or previous obesity ( 35.6% ) than in those who were continuously lean ( 24.3% ) ( p < 0.017 ) . in logistic regression analysis , previous obesity , as well as current obesity , was a significant risk factor for nephropathy , independent of sex , age , disease duration , hypertension , dyslipidemia , hba1c , and diabetic retinopathy . conclusions . obesity in the past , as well as the present body weight status , was a risk factor for diabetic nephropathy . linezolid is a synthetic antimicrobial agent of the oxazolidinone class with weak , nonspecific inhibitor of monoamine oxidase enzymes . concomitant therapy with an adrenergic or serotonergic agent or consuming tyramine ( > 100 mg / day ) may induce serotonin syndrome ( ss ) . we present a case report of near - fatal adverse interaction between linezolid and escitalopram inducing ss in a 65-year - old woman with sepsis , under empirical antibiotic treatment . this report also summarizes the current relevant literature as identified via pubmed , embase , and psycinfo , supplemented with a manual search of cross references .

aerobic ammonia oxidation , the first and rate - limiting step in nitrification , is the only biological process converting reduced to oxidized inorganic nitrogen species on earth . for over 100 years , this process was thought to be mediated by autotrophic beta - proteobacteria and gamma - proteobacteria ( aob ) occasionally supported by heterotrophic nitrifiers in soil environments . however , in situ measurements of nitrification in marine and terrestrial environments showed that ammonia oxidation often proceeds at substrate concentrations significantly below the growth threshold of cultured aob ( e.g. ) indicating the presence of unknown nitrifiers . the recent discovery of homologs of ammonia monooxygenase genes in archaea [ 57 ] and the cultivation of autotrophic ammonia - oxidizing archaea ( aoa ) [ 811 ] revealed that an additional group of microorganisms is able to catalyze this process . the widespread distribution of putative archaeal ammonia monooxygenase ( amo ) genes and their numerical dominance over their bacterial counterparts in most marine and terrestrial environments suggested that aoa play a major role in global nitrification [ 1215 ] , but our understanding of their evolutionary history and metabolic repertoire is still in its infancy . in 1992 , jed fuhrman 's team and ed delong reported the discovery of a novel clade of archaeal 16s rrna sequences from ocean surface waters , which formed a mesophilic sister group to the hyperthermohilic crenarchaeota . when it became apparent that this novel group contained aoa , these organisms were consequently also referred to as mesophilic crenarchaeota . this perception was questioned by phylogenetic analysis of the first available genome sequence of a putative aoa , the sponge symbiont candidatus cenarchaeum symbiosum . when brochier - armanet and colleagues analyzed a concatenated data set of 53 ribosomal proteins common to archaea and eukarya , they made the surprising observation that c. symbiosum branched off before the separation of crenarchaeota and euryarchaeota . based on this phylogenetic analysis , on gene presence / absence data , and on the diversity and wide distribution of aoa , they proposed that that these organisms belong to the phylum thaumarchaeota . recently , this analysis was extended to the ammonia - oxidizing candidatus nitrosopumilus maritimus , a marine group i.1a representative , and candidatus nitrososphaera gargensis , a soil group i.1b representative enriched from a hot spring . in this study , phylogenetic analysis of concatenated ribosomal proteins ( figure 1a ) and several other marker genes as well as presence / absence patterns of information processing machineries in archaea strongly supported the assignment of aoa to the deep - branching phylum thaumarchaeota . consistent with this finding , comparative genomics revealed that 6 conserved signature indels and > 250 proteins are unique to the thaumarchaeota c. symbiosum and n. pumilus and are not found in crenarchaeota . additional support for the phylum thaumarchaeota stems from comparative analysis of fosmid clones obtained from different deep - sea locations . among 200 phylogenetic trees of protein families present in thaumarchaeotal fosmids from these sites , thaumarchaeota sequences branched as separate cluster distinct from hyperthermophilic crenarchaeota and euryarchaeota in 162 phylogenetic trees . independent from genomic data , the presence of the lipid crenarchaeol in all analyzed aoa [ 9,2224 ] is consistent with a separate placement of these organisms in the archaeal tree as this lipid has so far not been found in any other bacterium or archaeon . thus , it seems likely that this membrane lipid , which may now be more appropriately termed thaumarchaeol , is an invention of an early thaumarchaote and represents a signature lipid for this phylum . revisiting the phylogenetic placement of thaumarchaeota in 16s rrna - based trees also reveals a clear separation from crenarchaeota and euryarchaeota ( figure 1b ) . a number of environmentally retrieved clone groups consisting of the sagmgc-1 group ( subsurface mine ) , group i.1c ( acidic soils ) , aloha group ( open ocean ) , psl12 group ( hot spring ) , and the hwcgiii / nitrosocaldus group ( hot springs / hydrothermal vents ) since this cluster is supported by all treeing methods and has a bootstrap value of 100% ( figure 1b ) , its representatives very likely all belong to the phylum thaumarchaeota and at least some of them might be aoa . supporting this hypothesis , a good correlation between copy numbers of archaeal amoa ( coding for the -subunit of ammonia monooxygenase ) and 16s rrna genes of the aloha group has been observed in the north pacific . it will be fascinating to see whether all thaumarchaeota have the capability to perform ammonia oxidation or whether certain members use a different energy metabolism . just recently two giant thaumarchaeota , candidatus giganthauma karukerense and candidatus giganthauma insulaporcus , were characterized by molecular methods but all attempts to amplify archaeal amoa genes failed . however , this could also be caused by primer bias as has been previously recognized for archaeal amoa - targeted surveys in deep ocean waters . currently , the mcg ( miscellaneous crenarchaeotic group ) , mbgb ( marine benthic group b ) , and hwcgi ( hot water crenarchaeotic group i ) clusters have no clear affiliation to any of the established archaeal phyla and show an unstable branching order when 16s rrna - based trees inferred with different treeing methods are compared ( figure 1b ) . little is known about these organisms but recently the first genome of a representative of the hwcgi cluster , that of candidatus caldiarchaeum subterraneum , was found to be distinct from other archaeal phyla including genes encoding a ubiquitin - like protein modifier system that was so far only found in eukaryotes . as a consequence , the lineage however , a comparative genome analysis by brochier - armanet and colleagues revealed some typical thaumarchaeal features in c. subterraneum and thus places it at the base of thaumarchaeota in protein trees ( for details see brochier - armanet et al . , this issue ) . with the availability of more genomes within this and related lineages , comparative genomics will show whether aigarchaeota represent a new archaeal phylum or will be classified as deep - branching members of the crenarchaeota or thaumarchaeota . the phylogenetic structure of aoa can also be analyzed by the functional marker gene amoa , which is found in all ammonia - oxidizing microorganisms . the presence of aoa within group i.1a and group i.1b thaumarchaeota as well as within the thaumarchaeota - group hwcgiii / nitrosocaldus is mirrored in the respective amoa phylogeny ( figure 1b ) . in addition , a fourth amoa - cluster with no established link to a thaumarchaeotal lineage in the 16s rrna - based tree became apparent during the accumulation of environmental amoa sequences within the last few years . since amoa sequences from a wide range of habitats ( including various marine , terrestrial , and hot water environments ) are affiliated with this lineage , we have named it the ubiquitous cluster. it is tempting to speculate that this cluster represents so - far unrecognized aoa within the sagmgc-1 , group i.1c , aloha , or psl12 cluster . almost every study that investigates ammonia - oxidizing thaumarchaeota uses the amoa gene to explore their diversity and abundance with the implicit assumption that all amoa - carrying archaea are oxidizing ammonia . however , of the > 10,000 deposited archaeal amoa sequences , thus far only four have been directly linked to archaeal strains for which experimental evidence of ammonia oxidation exists [ 811 ] . although phylogenetically closely related enzymes often perform the same function , it deserves consideration that the family of copper - containing membrane - bound monooxgenases ( cummo ) , to which archaeal ammonia monooxygenases belong , has a wide substrate range . in addition to ammonia [ ammonia monooxygenase ( amo ) in -proteobacteria , -proteobacteria , and thaumarchaeota ] , this includes methane [ particulate methane monooxygenase ( pmmo ) in -proteobacteria , -proteobacteria , verucomicrobia , and candidatus methylomirabilis oxyfera ] , and short - chained alkanes [ particulate butane monooxygenase ( pbmo ) in the gram - positive nocardioides strain cf8 ] . in addition , non - specific substrate catabolism such as oxidation of chlorinated ethenes and aromatic hydrocarbons has been observed with some members of this enzyme family , clearly indicating substrate promiscuity . therefore , it has been suggested that not necessarily the type of cummo but rather the downstream enzyme machinery defines the energy metabolism of a microorganism . for example , the -proteobacterium aob nitrosococcus oceani can oxidize methane but lacks all subsequent enzymes to gain energy by methane oxidation . likewise , co - oxidation of ammonia by methane oxidizing bacteria does not support their growth . furthermore , it is interesting to note that -proteobacterial amos are more closely related to -proteobacterial pmmos than to -proteobacterial amos and have a near equal substrate specificity for ammonia and methane . consequently , the mere presence of an amoa - like gene , transcript , or protein is insufficient to infer that the respective organism is oxidizing ammonia . currently , it is not clear whether aoa are strict autotrophs or also assimilate organic substrates . for n. maritimus , autotrophy has been shown and for n. gargensis co2-fixation has been experimentally demonstrated . incorporation of labeled bicarbonate into lipids , proteins , and cells of marine thaumarchaeota are consistent with autotrophy , which is enabled by a modified 3-hydroxypropionate/4-hydroxybutyrate ( hp / hb ) cycle for co2-fixation as found in known aoa genomes and in marine thaumarchaeal fosmids . however , analysis of the c. symbiosum and n. maritimus genomes as well as of thaumarchaeal fosmids from bathypelagic plankton also has revealed the presence of a tca cycle ( possibly incomplete ) and of potential transporters for organic substances such as amino acids , oligopeptides , and glycerol . thus , mixotrophic or even heterotrophic growth of marine thaumarchaeota as supported by other isotope labeling studies and natural distribution of radiocarbon in archaeal membrane lipids can to date not be excluded . furthermore , it has been suggested that parts of the hp / hb cycle can serve to co - assimilate organic compounds including , for example , 3-hydroxypropionate , an intermediate in the metabolism of the ubiquitous marine osmoprotectant dimethylsulphoniopropionate . for soil environments , co2-stable isotope probing revealed ammonia oxidizing activity of members of group i.1a as well as i.1b thaumarchaeota indicating an autotrophic or mixotrophic lifestyle . two of these studies found label incorporation into genes or transcripts of the 4-hydroxybutyryl - coa - dehydratase or acetyl - coa - propionyl - coa - carboxylase , respectively , with both enzymes being involved in the co2-fixing hp / hb cycle . however , growth of soil aoa with no concomitant incorporation of co2 has been also observed when nitrification was inhibited indicating that at least some soil aoa can grow heterotrophically . for comparison , heterotrophic growth of crenarchaeota that possess the hp / hb cycle is known for sulfolobus solfataricus and metallosphaera sedula with the latter being able to switch between an autotrophic and heterotrophic lifestyle . the question under which conditions aoa or aob dominate ammonia oxidation is currently attracting a lot of attention . for ammonia oxidation by the group i.1a thaumarchaeote n. maritimus , an extremely low substrate threshold ( < 10 nm total nh4 + nh3 , representing the detection limit of the used method ) and apparent km - value ( 133 nm ) were determined with the latter being very similar to in situ nitrification measurements made in oligotrophic oceans . adaptation to low ammonium concentrations has also been reported for the thermophilic group i.1b thaumarchaeote n. gargensis , indicating a widespread distribution of oligotrophic ammonia oxidation within the thaumarchaeota . in comparison , minimum total ammonium concentrations required for growth of cultured aob are 100-fold higher ( > 1 m near neutral ph ) with km - values ranging from 46 to 1780 m total ammonium . thus , a dominating activity of aoa in the large water bodies of oligotrophic oceans is highly likely with aob being restricted to organic - matter rich particles and coastal environments with higher nutrient loads . measured apparent km - values for soils range from 2 to 42 m total ammonium and may therefore be influenced by both aoa and aob . in general , activity of soil aoa was seen when total ammonia concentrations were below 15 g nh4-n ( g dw . soil ) whereas aob responded to high ammonia concentrations [ > 100 g nh4-n ( g dw . in addition , the form of supplied nitrogen might also play a critical role : aoa activity was seen when n was supplied as mineralized organic n derived from composted manure or soil organic matter and aob - dominated activity was seen with ammonia from inorganic fertilizer ( reviewed in ) . based on genome analyses of n. maritimus and c. symbiosum and due to the fact that aoa do not contain a homologue of the bacterial hydroxylamine oxidoreductase , a mechanism for ammonia oxidation distinctly different from that of aob has been proposed . here , ammonia is not oxidized via hydroxylamine ( nh2oh ) as in aob but rather via nitroxyl ( hno ) to nitrite , which possibly involves only 0.5 o2 per nh3 oxidized ( proposed by martin klotz ( louisville ) ) . this hypothesized lower oxygen demand could explain why aoa are found not only in fully areated soils and oxic marine waters but also in suboxic marine waters , sediments , and oxygen - depleted hot springs . in oxygen gradients of marine sediments and in the stratified water body of the black sea different aoa ecotypes aoa can also be found over a wide range of ph , temperature , salinity , and phosphate concentrations with some aoa being adapted to sulfidic environments , which extends the potential range of aoa niche differentiation to a multitude of environmental factors ( reviewed in ) . until recently , methanogenic euryarchaeota were the only known archaea of global relevance for element cycling . this perception changed with the discovery of ammonia - oxidizing archaea , which belong to the newly recognized archaeal phylum thaumarchaeota and contribute significantly to the global n - cycle and c - cycle . their shear abundance in the ocean ( up to 20% of all bacteria and archaea ) and extremely low substrate threshold for total ammonium provide compelling evidence for their role as dominant ammonia oxidizers in the open ocean , where they also contribute to primary production by their autotrophic ( or possibly partly mixotrophic ) lifestyle . the dominance of aoa over aob in many terrestrial environments can not be so easily explained . low km - values of unfertilized soils for ammonia oxidation might point to a contribution of certain aoa ecotypes to nitrification , especially under low ammonia availability . on the other hand , it is well possible that some soil thaumarchaeotes use other substrates than ammonia for energy generation and are heterotrophs or that they switch to ammonia oxidation only under certain environmental conditions . further dissection of the ecological interplay of aoa groups among themselves and with aob is urgently required and might reveal that aoa exhibit a similar type of niche partitioning as found for different nitrite oxidizers . here , prefer low nutrient and microoxic sites , with different nitrospira lineages adapted to different nitrite concentrations . papers of particular interest , published within the period of review , have been highlighted as: of special interest of outstanding interest of special interest of outstanding interest chronic obstructive pulmonary disease ( copd ) is a chronic respiratory disease characterized by a progressive decline in lung function and accompanied by respiratory symptoms , primarily dyspnea , cough , and sputum production.1,2 given the progressive nature of the disease , the aim of treatments is to reduce symptoms and exacerbations , thereby improving health - related quality of life . currently , the global initiative for chronic obstructive lung disease ( gold ) guidelines recommend initiation with a short - acting bronchodilator , followed by the addition of long - acting bronchodilators as symptoms and/or risk of exacerbations increase , and eventually adding inhaled steroids and/or roflumilast.2 commonly used bronchodilators include inhaled long - acting beta2-agonists ( labas ) ( eg , twice - daily formoterol , twice - daily salmeterol , and more recently once - daily indacaterol ) , inhaled long - acting antimuscarinic agents ( lamas ) ( eg , once - daily tiotropium , and recently twice - daily aclidinium and once - daily glycopyrronium ) , and oral methylxanthines ( eg , theophylline ) . bronchodilators improve lung function , reduce symptoms and exacerbations , and improve health - related quality of life.2 bronchodilators are frequently used in combination when higher effect sizes are warranted . in trials where a laba was administered in addition to a lama , the observed effect sizes were greater than those with either laba or lama alone,3,4 and in some cases the effects of the monotherapies have been shown to be additive when used in combination.5 however , effect sizes of bronchodilator monotherapies are not always additive , as seen in published trials of bronchodilator combinations . most recently , this was demonstrated with indacaterol , where the effect size of indacaterol administered with tiotropium compared with tiotropium monotherapy was much lower than the effect sizes observed in studies that compared indacaterol monotherapy with placebo.6 furthermore , more recent trials have found that monotherapy treatments have shown lower increases in forced expiratory volume in 1 second ( fev1 ) , compared to trials of the same investigational drug performed 5 or 10 years earlier . this may be due to evolution in trial designs , where , for example , later trials are more likely to permit concomitant treatment with other bronchodilators as part of usual care.7 additionally , there is evidence that the effect of bronchodilators varies depending on the severity of the patient s copd . patients with very severe copd show lower improvement of fev1 when treated with bronchodilators , compared with patients with moderate or severe copd.8,9 the laba olodaterol has been developed as a once - daily long - acting bronchodilator for maintenance treatment of copd . phase iii clinical trials of olodaterol have been completed , and the product , at the 5 mcg per day dose , has been approved in a number of european countries and canada , and is currently under regulatory review in the united states by the food and drug administration ( fda)10 and other regulatory agencies . to date , indacaterol is the only other once - daily laba approved and made available in many countries worldwide . indacaterol has been licensed in the united states and canada at a dose of 75 mcg per day.11,12 in most other countries , two doses are licensed : 150 mcg per day and 300 mcg per day for those patients for whom 150 mcg is not sufficient.13 in the absence of head - to - head , randomized controlled clinical trials ( rcts ) conducted on labas , the primary objective of this systematic literature review and meta - analysis was to provide estimates of relative efficacy of two new once - daily labas , olodaterol and indacaterol . a network meta - analysis was performed to indirectly compare the two treatments , linked through common treatment comparators . the review made two specific treatment comparisons : olodaterol 5 mcg compared with indacaterol 150 mcg , and olodaterol 5 mcg compared with indacaterol 75 mcg . a comprehensive and systematic literature review to identify rcts studying olodaterol and indacaterol in copd was undertaken to establish the evidence base for use in the meta - analyses . a systematic literature review was performed according to a prespecified protocol , to comply with the requirements of the national institute for health and care excellence single technology assessment.14 the following electronic databases were searched : 1 ) the cochrane library , including the cochrane database of systematic reviews , the cochrane central register of controlled trials , and the database of abstracts of reviews of effectiveness ; 2 ) medline and medline in - process ; and 3 ) embase . in addition , the following additional sources were searched to identify conference abstracts and published and unpublished studies : web sites for conference abstracts from the following organizations : american thoracic society ( 2010 and 2011 ) , european respiratory society ( 2009 and 2010 ) , and british thoracic society ( winter 2009 and winter 2010 ) ; bibliographic reference lists of the included studies and reviews ( searched for other relevant published studies ) ; trialtrove ( http://www.citeline.com/products/trialtrove/overview/ ) ; clinicaltrials.gov ( http://www.clinicaltrials.gov/ ) ; web sites for the fda and european medicines agency ; and the boehringer ingelheim trial database for olodaterol studies . the search strategies relied on three sets of terms : health condition of interest ( copd ) , study type ( rcts ) , and intervention ( olodaterol and/or indacaterol ) . appropriate terms were combined , and iterative searches were performed using other relevant terms and concepts . the searches were limited to articles published from 1 january 1990 , through 5 august 2011 , given the development timelines of the two compounds under study . articles in languages other than english were excluded . during systematic review and meta - analysis planning and execution , data for olodaterol were not publicly available , therefore , data on file with boehringer ingelheim were used . most data are now publicly available in the primary publications and/or on the fda web site , in the briefing documents for a pulmonary and allergy drug advisory committee meeting held in january 2013.10,1518 all data , including data not published in the pulmonary and allergy drug advisory committee document , are listed in the supplementary material . any disagreements were discussed with the research team to reach a consensus about study inclusion . during screening , further exclusions of studies were based on lack of outcome data , lack of a link into the treatment network , and study length ( ie , studies lasting less than 6 weeks ) . data extraction was performed by an experienced literature reviewer and quality checked by the statistician responsible for the meta - analyses . quality assessments were conducted for each included trial , using quality criteria recommended by the centre for reviews and dissemination.19 full details of this assessment can be found in the supplementary material . similar trough fev1 data at week 6 were permitted in the absence of week 12 data . other lung - function outcomes were considered for analysis , but trough fev1 was selected because it was the only outcome consistently reported across the evidence base . while the area under the fev1-time curve ( 03 hours post - dose ) was reported in all olodaterol trials , it was not reported for the indacaterol 150 or 75 mcg studies and so could not be included in the meta - analysis . st george s respiratory questionnaire ( sgrq ) total score was analyzed as change from baseline at week 12 . the proportion of responders based on sgrq total score was analyzed at week 12 , response was defined as a decrease ( improvement ) in sgrq total score of at least 4 points . transition dyspnea index ( tdi ) , by definition a change from baseline measure , was analyzed at week 12 . use of rescue medication ( eg , as - needed salbutamol ) was captured and analyzed as change from baseline in average number of puffs per day , using the maximum observed time for available data . finally , the proportion of patients experiencing at least one exacerbation , captured as a copd worsening adverse event , was analyzed for trials with a treatment duration of 24 weeks or longer reporting data for this outcome . some data imputations were required for standard errors , to maximize inclusion of change - from - baseline data into the meta - analyses . these imputations , together with further details on endpoint definitions are provided , where appropriate , in the data tables in the supplementary material . for network meta - analyses of dichotomous outcomes , a mixed log - binomial model was fit to estimate relative risks and confidence intervals . for network meta - analyses of continuous outcomes , a mixed normal - response model was fit to estimate mean differences and confidence intervals . these models included fixed treatment effects , fixed study effects , and random effects for the interaction between treatment and study . the random effects were included to allow the treatment effects to vary from study to study . the models were fit adopting an estimation approach using proc glimmix in sas version 9.2 ( sas institute inc . , cary , nc , usa ) and the methodology outlined by jones et al.20 in addition to network meta - analyses , direct meta - analyses were performed using standard techniques to assess heterogeneity within the network and to support the analysis that required use of the adjusted indirect comparison technique.2123 heterogeneity was investigated in several ways . for heterogeneity in trial designs , the a priori desire is to include a homogeneous set of trials in the network . to this end , trials were included / excluded based on permitted respiratory comedication during the trials ; patient subgroups of data were used where patient - level data were available ; and two additional indacaterol studies identified after completion of the systematic review were included,24 which allowed comparison of indacaterol with olodaterol when given with concomitant bronchodilation . for heterogeneity in analyses / results , the generalized chi - squared statistic divided by the remaining degrees of freedom ( should produce values close to 1.0 for a well - fitting model ) was calculated and heterogeneity tests for direct meta - analyses were performed where appropriate . a systematic literature review was performed according to a prespecified protocol , to comply with the requirements of the national institute for health and care excellence single technology assessment.14 the following electronic databases were searched : 1 ) the cochrane library , including the cochrane database of systematic reviews , the cochrane central register of controlled trials , and the database of abstracts of reviews of effectiveness ; 2 ) medline and medline in - process ; and 3 ) embase . in addition , the following additional sources were searched to identify conference abstracts and published and unpublished studies : web sites for conference abstracts from the following organizations : american thoracic society ( 2010 and 2011 ) , european respiratory society ( 2009 and 2010 ) , and british thoracic society ( winter 2009 and winter 2010 ) ; bibliographic reference lists of the included studies and reviews ( searched for other relevant published studies ) ; trialtrove ( http://www.citeline.com/products/trialtrove/overview/ ) ; clinicaltrials.gov ( http://www.clinicaltrials.gov/ ) ; web sites for the fda and european medicines agency ; and the boehringer ingelheim trial database for olodaterol studies . the search strategies relied on three sets of terms : health condition of interest ( copd ) , study type ( rcts ) , and intervention ( olodaterol and/or indacaterol ) . appropriate terms were combined , and iterative searches were performed using other relevant terms and concepts . the searches were limited to articles published from 1 january 1990 , through 5 august 2011 , given the development timelines of the two compounds under study . articles in languages other than english were excluded . during systematic review and meta - analysis planning and execution , data for olodaterol were not publicly available , therefore , data on file with boehringer ingelheim were used . most data are now publicly available in the primary publications and/or on the fda web site , in the briefing documents for a pulmonary and allergy drug advisory committee meeting held in january 2013.10,1518 all data , including data not published in the pulmonary and allergy drug advisory committee document , are listed in the supplementary material . any disagreements were discussed with the research team to reach a consensus about study inclusion . during screening , further exclusions of studies were based on lack of outcome data , lack of a link into the treatment network , and study length ( ie , studies lasting less than 6 weeks ) . data extraction was performed by an experienced literature reviewer and quality checked by the statistician responsible for the meta - analyses . quality assessments were conducted for each included trial , using quality criteria recommended by the centre for reviews and dissemination.19 full details of this assessment can be found in the supplementary material . similar trough fev1 data at week 6 were permitted in the absence of week 12 data . other lung - function outcomes were considered for analysis , but trough fev1 was selected because it was the only outcome consistently reported across the evidence base . while the area under the fev1-time curve ( 03 hours post - dose ) was reported in all olodaterol trials , it was not reported for the indacaterol 150 or 75 mcg studies and so could not be included in the meta - analysis . st george s respiratory questionnaire ( sgrq ) total score was analyzed as change from baseline at week 12 . the proportion of responders based on sgrq total score was analyzed at week 12 , response was defined as a decrease ( improvement ) in sgrq total score of at least 4 points . transition dyspnea index ( tdi ) , by definition a change from baseline measure , was analyzed at week 12 . use of rescue medication ( eg , as - needed salbutamol ) was captured and analyzed as change from baseline in average number of puffs per day , using the maximum observed time for available data . finally , the proportion of patients experiencing at least one exacerbation , captured as a copd worsening adverse event , was analyzed for trials with a treatment duration of 24 weeks or longer reporting data for this outcome . some data imputations were required for standard errors , to maximize inclusion of change - from - baseline data into the meta - analyses . these imputations , together with further details on endpoint definitions are provided , where appropriate , in the data tables in the supplementary material . for network meta - analyses of dichotomous outcomes , a mixed log - binomial model was fit to estimate relative risks and confidence intervals . for network meta - analyses of continuous outcomes , a mixed normal - response model was fit to estimate mean differences and confidence intervals . these models included fixed treatment effects , fixed study effects , and random effects for the interaction between treatment and study . the random effects were included to allow the treatment effects to vary from study to study . the models were fit adopting an estimation approach using proc glimmix in sas version 9.2 ( sas institute inc . , cary , nc , usa ) and the methodology outlined by jones et al.20 in addition to network meta - analyses , direct meta - analyses were performed using standard techniques to assess heterogeneity within the network and to support the analysis that required use of the adjusted indirect comparison technique.2123 heterogeneity was investigated in several ways . for heterogeneity in trial designs , the a priori desire is to include a homogeneous set of trials in the network . to this end , trials were included / excluded based on permitted respiratory comedication during the trials ; patient subgroups of data were used where patient - level data were available ; and two additional indacaterol studies identified after completion of the systematic review were included,24 which allowed comparison of indacaterol with olodaterol when given with concomitant bronchodilation . for heterogeneity in analyses / results , the generalized chi - squared statistic divided by the remaining degrees of freedom ( should produce values close to 1.0 for a well - fitting model ) was calculated and heterogeneity tests for direct meta - analyses were performed where appropriate . a total of 142 titles were retrieved through database searches and a further 31 through other sources such as conference web sites . after removal of duplicates , 110 titles and abstracts were screened . after applying the screening inclusion and exclusion criteria to both the titles / abstracts and the full - text articles , 23 publications , which reported data from a total of ten indacaterol trials ( b1302,25 b2201,26 study b2354,27 study b2355,28 inhance,29 inlight-1,30 inlight-2,31 insist,32 intensity,33 and involve34 ) , were eligible for meta - analysis , outlined in the prisma diagram in figure 1.35 from the boehringer ingelheim trial database search , eight olodaterol trials were identified that met the inclusion criteria ( study numbers 1222.11 [ nct00782210],15 1222.12 [ nct00782509],15 1222.13 [ nct00793624],16 1222.14 [ nct00796653],16 1222.24 [ nct00931385],17 1222.25 [ nct00932646],17 1222.39 [ nct01040689],18 and 1222.40 [ nct01040728]).18 during the meta - analysis phase of the project , two further publications were identified and included in the evidence base.24,36 cope et al36 presented a network meta - analysis of four indacaterol trials ( involve , inhance , inlight-1 , and inlight-2 ) already included in the evidence base . mahler et al24 presented results for two indacaterol trials ( intrust-1 and intrust-2 ) that were not otherwise identified at the time of the literature searches . table 1 lists the 20 included studies ( 18 from the systematic review and 2 identified after the review ) and provides details on the included treatments , sample sizes , concomitant bronchodilator use , and whether the trials permitted patients with very severe copd ( gold stage iv).37 further trial / baseline characteristics are presented in the supplementary material . the supplementary material also presents data for each of the outcomes for analysis and the descriptions of how the data were captured from each trial . the resulting treatment network for this evidence base is presented in the supplementary material ( figure a1 ) . the different doses of olodaterol and indacaterol were considered as unique entities in this network and in these meta - analyses . figure 2 presents an abbreviated version of the complete network and highlights the trials contributing to the indirect comparisons of primary interest ( ie , the comparison of olodaterol 5 mcg with indacaterol 75 mcg and with indacaterol 150 mcg ) . an important difference among the studies included in the evidence base is whether concomitant use of noninvestigational copd treatments was permitted in the trials by design . specifically , the olodaterol trials allowed concomitant use of lamas ( tiotropium ) , short - acting muscarinic antagonists ( samas ) ( ipratropium ) , inhaled corticosteroids , and xanthines ( theophylline ) . four of the eight included olodaterol trials used stratified randomization according to lama use to ensure a balance in tiotropium users across treatment arms . two olodaterol trials did not permit concomitant lama treatment , because tiotropium was one of the investigational drugs . none of the indacaterol trials originally identified in the systematic literature review permitted any concomitant bronchodilator use ( ie , samas and lamas were not allowed ) or xanthine use ; all of the indacaterol trials permitted use of inhaled corticosteroids . in contrast , the two intrust trials required the co - administration of indacaterol with tiotropium;24 thus , 100% of patients in those trials received tiotropium in addition to indacaterol or placebo ( inhaled steroids were also allowed , but not xanthines ) . as - needed short - acting beta - agonists were permitted as rescue medication in all olodaterol and indacaterol trials , and their use was captured via the rescue medication outcome . in addition , there were differences in the patient populations tested in the trials ; while the olodaterol trials included patients with moderate to very severe copd ( gold ii to gold iv),37 the indacaterol trials included only patients with moderate to severe copd ( ie , excluded patients with very severe copd [ gold stage iv]).37 given the findings from the trial characteristics summary ( ie , to take into account heterogeneity in trial design , specifically concomitant bronchodilator use ) , two analyses were performed using subgroups of trials or patients that could be considered to be subject to similar trial conditions and therefore comparable . given the data availability , these analyses could only be performed for the change from baseline in trough fev1 at week 12 . lama - free therapy and included all trials in which patients did not receive concomitant lama ( ie , all indacaterol trials identified in the original systematic review and the olodaterol trials 1222.39 and 1222.40 ) . lama add - on therapy and included all trials / subgroups in which all patients received concomitant or co - administered lama ( ie , stratified subgroups of olodaterol trials 1222.11 , 1222.12 , 1222.13 , and 1222.14 ; intrust trials for indacaterol ) . because intrust had only placebo as a common comparator , direct meta - analysis and the adjusted indirect comparison technique were used for this analysis . a third analysis population incorporated all trials ( full network ) identified by the systematic review and was considered a sensitivity analysis for the trough fev1 outcome . given data availability , this population was the only possible analysis population for the other outcomes . change from baseline in trough fev1 for each of the analysis populations . for the lama - free analysis , which included only trials explicitly excluding concomitant use of lama treatment , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . for the lama add - on analysis , which included only trials / subgroups receiving either concomitant or co - administered lama treatment , no differences were seen between olodaterol 5 mcg and indacaterol 150 mcg . no data were available for indacaterol 75 mcg for the lama add - on analysis . for the sensitivity analysis of the full network , the indirect comparisons favored indacaterol , statistically significantly for the 150 mcg dose . figure 4 presents the indirect treatment comparison results for the continuous outcomes tdi , change from baseline in sgrq total score , and change from baseline in rescue medication puffs per day for the full network analysis . despite the known heterogeneity among the trials included in the full network , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . figure 5 presents the indirect treatment comparison results for the dichotomous outcomes sgrq response and proportion of patients with exacerbations for the full network analysis . again , despite the known heterogeneity among the trials , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg , where data were available . as shown in the separate analyses for change from baseline in trough fev1 , the identified heterogeneity in trial design with respect to concomitant bronchodilator use impacted the results and conclusions of the meta - analyses . table 3 presents generalized chi - squared divided by the remaining degrees of freedom statistics for each network meta - analysis . because the estimates were all fairly close to 1 , the models could be assumed to fit the data well , but this does not eradicate the issues of known trial heterogeneity . table 4 presents direct meta - analyses heterogeneity test results for comparisons with placebo ( where possible ) . these estimates do not display significant and consistent heterogeneity of trial results within each arm of the network diagram , but it should be noted that such heterogeneity tests can lack statistical power with small trial numbers . additionally , information regarding the consistency assumption within the network as a whole is not provided due to the lack of head - to - head data between the selected treatments of interest . forest plots for the direct meta - analyses are presented in the supplementary material ( figure a2 through figure a21 ) . a total of 142 titles were retrieved through database searches and a further 31 through other sources such as conference web sites . after removal of duplicates , 110 titles and abstracts were screened . after applying the screening inclusion and exclusion criteria to both the titles / abstracts and the full - text articles , 23 publications , which reported data from a total of ten indacaterol trials ( b1302,25 b2201,26 study b2354,27 study b2355,28 inhance,29 inlight-1,30 inlight-2,31 insist,32 intensity,33 and involve34 ) , were eligible for meta - analysis , outlined in the prisma diagram in figure 1.35 from the boehringer ingelheim trial database search , eight olodaterol trials were identified that met the inclusion criteria ( study numbers 1222.11 [ nct00782210],15 1222.12 [ nct00782509],15 1222.13 [ nct00793624],16 1222.14 [ nct00796653],16 1222.24 [ nct00931385],17 1222.25 [ nct00932646],17 1222.39 [ nct01040689],18 and 1222.40 [ nct01040728]).18 during the meta - analysis phase of the project , two further publications were identified and included in the evidence base.24,36 cope et al36 presented a network meta - analysis of four indacaterol trials ( involve , inhance , inlight-1 , and inlight-2 ) already included in the evidence base . mahler et al24 presented results for two indacaterol trials ( intrust-1 and intrust-2 ) that were not otherwise identified at the time of the literature searches . table 1 lists the 20 included studies ( 18 from the systematic review and 2 identified after the review ) and provides details on the included treatments , sample sizes , concomitant bronchodilator use , and whether the trials permitted patients with very severe copd ( gold stage iv).37 further trial / baseline characteristics are presented in the supplementary material . the supplementary material also presents data for each of the outcomes for analysis and the descriptions of how the data were captured from each trial . the resulting treatment network for this evidence base is presented in the supplementary material ( figure a1 ) . the different doses of olodaterol and indacaterol were considered as unique entities in this network and in these meta - analyses . figure 2 presents an abbreviated version of the complete network and highlights the trials contributing to the indirect comparisons of primary interest ( ie , the comparison of olodaterol 5 mcg with indacaterol 75 mcg and with indacaterol 150 mcg ) . an important difference among the studies included in the evidence base is whether concomitant use of noninvestigational copd treatments was permitted in the trials by design . specifically , the olodaterol trials allowed concomitant use of lamas ( tiotropium ) , short - acting muscarinic antagonists ( samas ) ( ipratropium ) , inhaled corticosteroids , and xanthines ( theophylline ) . four of the eight included olodaterol trials used stratified randomization according to lama use to ensure a balance in tiotropium users across treatment arms . two olodaterol trials did not permit concomitant lama treatment , because tiotropium was one of the investigational drugs . none of the indacaterol trials originally identified in the systematic literature review permitted any concomitant bronchodilator use ( ie , samas and lamas were not allowed ) or xanthine use ; all of the indacaterol trials permitted use of inhaled corticosteroids . in contrast , the two intrust trials required the co - administration of indacaterol with tiotropium;24 thus , 100% of patients in those trials received tiotropium in addition to indacaterol or placebo ( inhaled steroids were also allowed , but not xanthines ) . as - needed short - acting beta - agonists were permitted as rescue medication in all olodaterol and indacaterol trials , and their use was captured via the rescue medication outcome . in addition , there were differences in the patient populations tested in the trials ; while the olodaterol trials included patients with moderate to very severe copd ( gold ii to gold iv),37 the indacaterol trials included only patients with moderate to severe copd ( ie , excluded patients with very severe copd [ gold stage iv]).37 given the findings from the trial characteristics summary ( ie , to take into account heterogeneity in trial design , specifically concomitant bronchodilator use ) , two analyses were performed using subgroups of trials or patients that could be considered to be subject to similar trial conditions and therefore comparable . given the data availability , these analyses could only be performed for the change from baseline in trough fev1 at week 12 . lama - free therapy and included all trials in which patients did not receive concomitant lama ( ie , all indacaterol trials identified in the original systematic review and the olodaterol trials 1222.39 and 1222.40 ) . the second analysis population was lama add - on therapy and included all trials / subgroups in which all patients received concomitant or co - administered lama ( ie , stratified subgroups of olodaterol trials 1222.11 , 1222.12 , 1222.13 , and 1222.14 ; intrust trials for indacaterol ) . because intrust had only placebo as a common comparator , direct meta - analysis and the adjusted indirect comparison technique were used for this analysis . a third analysis population incorporated all trials ( full network ) identified by the systematic review and was considered a sensitivity analysis for the trough fev1 outcome . given data availability , this population was the only possible analysis population for the other outcomes . figure 3 presents the indirect treatment comparison results for the outcome change from baseline in trough fev1 for each of the analysis populations . for the lama - free analysis , which included only trials explicitly excluding concomitant use of lama treatment , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . for the lama add - on analysis , which included only trials / subgroups receiving either concomitant or co - administered lama treatment , no data were available for indacaterol 75 mcg for the lama add - on analysis . for the sensitivity analysis of the full network , the indirect comparisons favored indacaterol , statistically significantly for the 150 mcg dose . figure 4 presents the indirect treatment comparison results for the continuous outcomes tdi , change from baseline in sgrq total score , and change from baseline in rescue medication puffs per day for the full network analysis . despite the known heterogeneity among the trials included in the full network , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg . figure 5 presents the indirect treatment comparison results for the dichotomous outcomes sgrq response and proportion of patients with exacerbations for the full network analysis . again , despite the known heterogeneity among the trials , no differences were seen between olodaterol 5 mcg and either indacaterol 75 mcg or indacaterol 150 mcg , where data were available . as shown in the separate analyses for change from baseline in trough fev1 , the identified heterogeneity in trial design with respect to concomitant bronchodilator use impacted the results and conclusions of the meta - analyses . table 3 presents generalized chi - squared divided by the remaining degrees of freedom statistics for each network meta - analysis . because the estimates were all fairly close to 1 , the models could be assumed to fit the data well , but this does not eradicate the issues of known trial heterogeneity . table 4 presents direct meta - analyses heterogeneity test results for comparisons with placebo ( where possible ) . these estimates do not display significant and consistent heterogeneity of trial results within each arm of the network diagram , but it should be noted that such heterogeneity tests can lack statistical power with small trial numbers . additionally , information regarding the consistency assumption within the network as a whole is not provided due to the lack of head - to - head data between the selected treatments of interest . forest plots for the direct meta - analyses are presented in the supplementary material ( figure a2 through figure a21 ) . in the absence of a direct head - to - head comparison , indirect comparisons can provide exploratory insights into the relative effectiveness of olodaterol and indacaterol . the current study indirectly compares olodaterol and indacaterol , while accounting for the considerable systematic heterogeneity in trial design among olodaterol and indacaterol trials . evaluating lung function , trials / subgroups were selected in order to form indirect treatment comparisons based on patients subject to similar trial conditions , in which no evident differences between olodaterol and indacaterol were found . there were important differences among the olodaterol trials and indacaterol trials with respect to allowed concomitant copd medications . although all trials allowed concomitant inhaled steroid use and allowed the use of short - acting beta - agonists as rescue medication on an as - needed basis , the olodaterol trials also allowed concomitant use of lamas ( tiotropium ) , samas ( ipratropium ) , and xanthines ( theophylline ) , whereas the indacaterol trials did not . another notable difference was that olodaterol trials enrolled patients across the full disease severity spectrum , from moderate to very severe copd ( gold ii - iv),37 whereas the indacaterol trials were restricted to patients with moderate to severe copd ( gold ii / iii).37 this difference in patient populations , and therefore potential lung function response,8 could not be addressed in this meta - analysis because olodaterol trials were not stratified for disease severity and therefore using the subgroup excluding gold iv patients for the meta - analysis would not be a randomized comparison . the comparative efficacy of indacaterol and olodaterol differed depending on the subset of trials and patients included in the analyses . change from baseline in trough fev1 was similar among treatments when analyzing trials or data deemed to be comparable . in contrast , analyses not taking into consideration differences in allowed concomitant therapies can lead to inappropriate conclusions from the indirect comparisons . thus , selecting studies of similar design for the drugs of interest is important in order to estimate the true differences in effect size . acknowledging the known differences of the patient populations recruited to the trials ( very severe copd patients included in trials of olodaterol , but not indacaterol ) , it is interesting to note that the absolute difference seen in the sensitivity analysis for trough fev1 outcome is not replicated in either sgrq endpoint evaluating health - related quality of life , where there are no differences among the treatments , or in the other endpoints investigated ( exacerbations , rescue medication use , and tdi ) . assessing the results for external validity with previously published meta - analyses,36,3841 the direct meta - analyses matched closely with the estimated treatment effects for indacaterol ; however , because olodaterol data have only recently been published , the indirect comparisons have not been externally validated . the smaller effect sizes of laba administered in addition to a lama , compared with the effect sizes of a laba alone , were replicated . specifically , for indacaterol 150 mcg versus placebo , the effect size for trough fev1 change from baseline was 0.07 liters when studied in combination with concomitant lama ( supplementary material , figure a6 ) , and 0.17 liters when studied in the absence of concomitant lama ( supplementary material , figure a4 ) . as with all meta - analyses , certain limitations should be considered when interpreting the results of this meta - analysis . some between - trial differences in inclusion and exclusion criteria that influence the meta - analytic results were identified , especially with regard to the use of concomitant respiratory medication . the olodaterol trials could be considered to be studying treatment effects likely to be seen in real - world settings , whereas the indacaterol trials provide an estimate of absolute treatment effect . a limitation in the lama - add on analysis of the lung - function endpoint is that only add - on tiotropium , and no other important bronchodilators ( eg , samas or xanthines ) , could be investigated , thus limiting the generalizability of the findings . another limitation was that the add - on tiotropium in the olodaterol trials was concomitant medication , whereas in the indacaterol trials it was part of the investigational treatment . this limitation could lead to differences in application of , monitoring of , and compliance with the treatment . a limitation with regard to the lama - free analysis for the lung - function endpoint was that the included olodaterol trials were cross - over trials lasting 6 weeks , whereas the indacaterol trials were parallel - group trials lasting 12 weeks . however , this trial heterogeneity may not influence the relative treatment effects and therefore indirect comparison results , because the relative treatment differences are maintained between week 6 and week 12 , based on studies comparing olodaterol 5 mcg and formoterol 12 mcg.10,1518 finally , analysis of endpoints other than change from baseline in trough fev1 were possible only on the evidence base of trials that are inherently dissimilar with respect to concomitant bronchodilator use . thus , these analyses should be interpreted with caution . in conclusion , based on the analyses of change from baseline in trough fev1 , when compared under similar trial conditions , olodaterol 5 mcg and indacaterol 75 mcg or 150 mcg seem to be equally effective in the treatment of patients with copd . only head - to - head studies would be able to confirm the equal effectiveness of olodaterol and indacaterol . this research highlights the importance of concomitant copd medication and study population when estimating treatment effects in copd clinical trials . mr roskell is a full - time employee of bresmed health solutions , but performed this work while employed by rti health solutions . he has the following conflicts of interest : he is a member of the gold scientific committees and supports the implementation of the gold guidelines recommendations . he has also received honoraria , consulting fees , and advisory board and speaking fees from bayer , pfizer , glaxosmithkline , boehringer ingelheim , and forest laboratories . he is the principal investigator for research grants at the university of texas health science center at san antonio and was paid for participating in multicenter clinical trials sponsored by glaxosmithkline and the national institutes of health . dr hamilton , dr disse , and dr becker are full - time employees of boehringer ingelheim gmbh .

thaumarchaeota range among the most abundant archaea on earth . initially classified as mesophilic crenarchaeota , comparative genomics has recently revealed that they form a separate and deep - branching phylum within the archaea . this novel phylum comprises in 16s rrna gene trees not only all known archaeal ammonia oxidizers but also several clusters of environmental sequences representing microorganisms with unknown energy metabolism . ecophysiological studies of ammonia - oxidizing thaumarchaeota suggest adaptation to low ammonia concentrations and an autotrophic or possibly mixotrophic lifestyle . extrapolating from the wide substrate range of copper - containing membrane - bound monooxygenases , to which the thaumarchaeal ammonia monooxygenases belong , the use of substrates other than ammonia for generating energy by some members of the thaumarchaeota seems likely . purposein the absence of head - to - head clinical trials comparing the once - daily , long - acting beta2-agonists olodaterol and indacaterol for the treatment of chronic obstructive pulmonary disease ( copd ) , an indirect treatment comparison by systematic review and synthesis of the available clinical evidence was conducted.methodsa systematic literature review of randomized , controlled clinical trials in patients with copd was performed to evaluate the efficacy and safety of olodaterol and indacaterol . network meta - analysis and adjusted indirect comparison methods were employed to evaluate treatment efficacy , using outcomes based on trough forced expiratory volume in 1 second ( fev1 ) , transition dyspnea index , st george s respiratory questionnaire total score and response , rescue medication use , and proportion of patients with exacerbations.resultseighteen trials were identified for meta - analysis ( eight , olodaterol ; ten , indacaterol ) . olodaterol trials included patients of all severities , whilst indacaterol trials excluded patients with very severe copd . concomitant maintenance bronchodilator use was allowed in most olodaterol trials , but not in indacaterol trials . when similarly designed trials / data were analyzed for change from baseline in trough fev1 ( liters ) , the following mean differences ( 95% confidence interval ) were observed : trials excluding concomitant bronchodilator : indacaterol 75 mcg versus olodaterol 5 mcg , 0.005 ( 0.077 to 0.067 ) , and indacaterol 150 mcg versus olodaterol 5 mcg , 0.020 ( 0.036 to 0.077 ) ; trials with concomitant tiotropium : indacaterol 150 mcg versus olodaterol 5 mcg , 0.000 ( 0.043 to 0.042 ) . in sensitivity analyses of the full network , results for change from baseline in trough fev1 favored indacaterol , but this dataset suffered from trial design heterogeneity . for the other endpoints investigated , no statistically significant differences were found when analyzed in the full network.conclusionwhen compared under similar trial conditions , olodaterol and indacaterol have similar efficacy in patients with copd . this research highlights the importance of considering the concomitant copd medication when evaluating treatment effects in copd .

diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion , insulin action , or both ( 1 ) . the chronic hyperglycemia of diabetes is associated with long - term damage , dysfunction , and failure of different organs , especially of eyes , kidneys , nerves , heart , and blood vessels ( 1 ) . the main forms of diabetes are divided into those caused by lack of insulin secretion , due to damage of - cells of the pancreas ( type 1 dm ) , and those that are a consequence of insulin resistance that occurs at the level of skeletal muscles , liver and adipose tissue , with varying degrees of - cells damage ( type 2 dm ) . diabetes mellitus classification ( 2 ) the prevalence of type 2 diabetes is increasing globally and represents a heavy burden on public health and socioeconomic development of all nations ( 3 ) . type 2 diabetes is a multifactorial disease and due to a combination of environmental and genetic risk factors ( many environmental risk factors contribute to the pathogenesis of type 2 diabetes , including lifestyles such as sedentary behavior , diet , smoking and alcohol consumption , internal environmental factors such as inflammatory factors , adipocytokines and hepatocyte factors , external environmental factors such as environmental endocrine disruptors ) ( 3 ) . so no isolated known defect predominates , as is the case with hla connection with type 1 dm . . it was found that the risks for diabetes in african - americans , hispanics , and native americans are approximately 2 , 2.5 , and 5 times greater , respectively , than in caucasians . the main reason for this is that symptoms , when seen on their own , seem harmless . however , the earlier diabetes is diagnosed , the greater the chances are that serious complications , which can result from having diabetes , can be avoided . most common diabetes symptoms are : frequent urination , disproportionate thirst , intense hunger , weight gain , unusual weight loss , increased fatigue , irritability , blurred vision , cuts and bruises do not heal properly or quickly , skin and/or yeast infections , itchy skin , gums are red and/or swollen gums pulled away from teeth , frequent gum disease / infection , sexual dysfunction among men , numbness or tingling , especially in feet and hands . the aim of this paper was to evaluate questionnaires on the assessment of risk factors for diabetes mellitus type 2 in order to get information about risk of population for getting diabetes mellitus type 2 on the territory of canton sarajevo ( bosnia and herzegovina ) . a total of 540 questionnaires handed out randomly to citizens of canton sarajevo of all ages , sexes and educational levels ( in january 2016 ) were analyzed . in a survey conducted by the students of medical faculty , university of sarajevo a total of 540 people were questioned . out of that number the research was done in canton sarajevo in four municipalities : center ( 46% of questionnaires ) , novo sarajevo ( 20% ) , novi grad ( 27% ) and stari grad ( 7% ) . in determining the age of the examinees there were five categories used , the first one being the group of young people under the age of 35 years and the last group categorized as elder people with the age over 64 years ( bellow 35 years 42% , 35 - 44 years 16% , 45 - 54 years 18% , 55 - 64 years 15% , over 64 years 9% ) . in determining the height of the examinees , the answers were of an open type , so considering all the given numbers a classification has been made in total of 8 categories with a range of 5 cm in between . the first category included the height under 150 cm and the last with height over 186 cm ( the largest number of respondents , 30% of them were in the range of 170 - 175 cm ) . the weight of the patients was also of an open type , and after all the given number there has been a classification with a total of six categories , with a range of 10 kg , the first one considering all the values under 49 kg and the last all the values above 90 kg . the largest number of respondents were in the range 70 - 79 kg ( 32% ) . in the survey while asking the examinee of their body mass index ( bmi ) , this number was only approximate considering the height and weight of the examinee and was nt taken in a professional manner . bmi index of examinees after evaluating these basic information on phenotype characteristics of the examinees , the next group of questions referred to risk factors on diabetes type 2 and it s possible manifestation . did any of your close relatives have diabetes is the basic information in every family history which can roughly show us the history of this disease amongst the closest relatives of our examinee ( figure 2 ) . family history of examinees our further questions were related to the risk factors concerning the environment but which have the effect on the progress of diabetes . these questions are as follows : do you have at least 30 minutes of physical exercise daily ( including regular daily activities ) and how often do you eat fruit , vegetables , grains . the question : do you suffer from high blood pressure is a manifestation of some other chronic disease for which the examinee also has got or not genetic predispositions . risk environment factors was a high blood sugar level ever detected earlier ( e.g. during regular testing in pregnancy or during other illness ) is a question from the patients history and can also roughly show us the history of blood sugar levels in our patients ( answer yes was given from 35% of examinees ) . the last question : do you have any of the listed symptoms ( fatigue , paleness , excessive sweating , heart thumping , shaking , reduced vision , itching , often infections , cramps in legs , problems with concentration , excessive hunger , dry mouth and excessive thirst , excessive urinating , urinating at night ) is a question whether or not there are already visible manifestations of diabetes and are there chronic illnesses which may imply on further progress of diabetes type two . in the first group of symptoms we have : paleness , fatigue , excessive sweating , heart thumping , shaking , reduced vision , itching , often infections , cramps in legs , problems with concentration , excessive hunger and dry mouth . in the other group of symptoms we have : excessive thirst , excessive urinating , urinating at night ( figure 4 ) . the fact is that by development of bosnia and herzegovina and approaching european standards and norms , the awareness of the disease is growing , and also awareness of the importance of therapy including pharmacological and lifestyle changes . at the end after finalizing the points that each question carried and after finalizing the risk factor for the onset of diabetes type two in the next ten years , a total of five categories was used to describe these probabilities ( table 2 , figure 5 ) . risk groups of patients the amount of risk for the onset of diabetes type two in the next ten years diabetes disease rate of the population of the federation of bosnia and herzegovina , 20092013rate/10.000 inhabitants ( 9 ) type ii dm patients generally carry a number of risk factors for cvd , including hyperglycemia , abnormal lipid profiles , alterations in inflammatory mediators and coagulation / thrombolytic parameters , as well as other nontraditional risk factors , many of which may be closely associated with insulin resistance . therefore , successful management of cvd associated with diabetes represents a major challenge to the clinicians . the japanese trial included 458 men randomized to receive either intensive lifestyle intervention ( n = 102 ) or standard intervention ( n = 356 ) . the aims of the intensive intervention were body weight reduction if bmi was 22 kg / m2 , to consume large amounts of vegetables while reducing the amount of other foods by 10% , reduction of fat ( < 50 g / day ) and alcohol intake ( < 50 g / day ) , and physical activity > 3040 min / day , and result was that 4-year incidence of type 2 diabetes in the intervention group was 67% lower than in the control group ( 6 ) . number of people living with diabetes is expected to rise from 366 million in 2011 to 552 million by the year 2030 , if no urgent action is taken ( this equates to approximately three new cases every ten seconds or almost ten million per year ) ( 7 ) . one fifth of all adults with diabetes live in the south - east asia region ( 8) . the results of studies on the state of health of population in the federation of bosnia and herzegovina have shown that over fifth of the population older than 18 years ( 21.7% ) have value of blood glucose equal or higher than 6.1 mmol / l . ( 9 ) the rate of the population with diabetes in the federation is continuously growing and the number of diseased is considered to be undervalued because federation of bosnia and herzegovina has not established a register of people with diabetes , but the data is not comprehensive and monitoring of indicators is not enough . the fact is that this is a major problem , and also it burdens already fragile health care system in bosnia and herzegovina ( it is estimated that in bosnia and herzegovina in year 2011 , patient with diabetes gives an average of 629 usd for the treatment of diabetes and its complications , and such funds , by development of more expensive drugs , are growing from year to year ) . this is corroborated by the results of study on the state of health of the population in the federation of b&h , in which 9.6% of the population older than 18 years reported that a doctor at any time in their life diagnosed diabetes ( 9 ) . in the federation of bosnia and herzegovina , according to estimates of the international diabetes federation ( idf ) , over 166,000 persons have diabetes , or about 9.2% of adult population . this percentage will grow significantly unless decisive and systematic measures on suppression for this epidemic of modern times are taken . establish a registry of people with diabetes in the fb&h should be one of the main goals of the health system , and after that the monitoring or continuous collection of data on the disease . diabetes is a social disease because it is massive and chronic , as its treatment is expensive and lifelong , which reduces the ability to work and shortens working life of the diseased , which makes it difficult to maintain social relations of the diseased in the family , school , workplace and environment . the emergence of first chronic complications in patients creates uncertainty , which is transmitted to the family and work environment ( 10 ) . this increases already high cost of treatment of these patients and sometimes beyond economic opportunities and family health insurance . consequently , the prevention of type ii dm is imperative especially in primary health care . as seen earlier ( figure 4 ) there is a relatively low risk of getting diabetes in the next ten years in the majority of the population . these results are rather encouraging but may in some way be in confrontation with the statistics which show a rapid outburst of diabetes . the life - style is the main reason for such a thing to happen , and looking at theses questionnaires , we might get the feeling that we really do live in a , conditionally speaking , physically active society . however , good attitude and knowledge about diabetes amongst society is present which is good news and derives from all the media campaign and promotion programs in hand . this study has a lot of limitations , but it would be wise to continue doing research on this topic , perhaps making the next studies more complex , ruling out the main reasons for the onset amongst these already listed risk factors , statistically calculating each variable , and making these questionnaires more complex as well as including other means of information gathering , such as interviews , reviewing medical records etc . acute right heart syndrome ( arhs ) may be defined as sudden deterioration in the right ventricular ( rv ) function and failure of the rv of the heart to deliver adequate blood flow to the pulmonary circulation , resulting in systemic hypoperfusion . in the context of critical illness evidence of central venous pressure ( cvp ) overload in conjunction with rv contractile dysfunction , is usually present in arhs . we searched pubmed , embase , cochrane library and google scholar , for articles reporting on rv dysfunction and failure . the relevant papers were extracted in full and references from extracted papers were checked for any additional relevant articles . an overview of the arhs pathophysiology , diagnostic tools for the assessment of the acutely failing rv in critical illness and measures including vasoactive agents , ventilatory strategies and mechanical support is provided in the current paper . the main functions of the rv are : a ) maintenance of adequate pulmonary perfusion pressure in order to deliver desaturated mixed venous blood to the respiratory membrane ; b ) maintenance of low systemic venous pressure in order to prevent organ congestion . the rv is anatomically adapted for the generation of low - pressure perfusion and it is very sensitive to changes in afterload . . the differences between the structure and function of the rv compared to the left ventricle ( lv ) are outlined in table 1 and figure 1 compares the pressure - volume ( p - v ) loop of the rv with that of the lv . little time is spent in isovolumetric contraction , giving a triangular - shaped rv p - v loop , in contrast to the almost square loop of the lv ( 25 ) . the rv in critical illness arhs is not necessarily associated with an increase in pulmonary vascular resistance ( pvr ) and pulmonary arterial hypertension ( pah ) . the syndrome can be due to rv pressure / volume overload or rv contractile dysfunction . consequence is low cardiac output ( co ) with low mean arterial pressure ( map ) , exacerbating rv dysfunction . rv failure begets rv failure leading to a progressive downward spiral of worsening ischemia , myocardial dysfunction and shock . in mechanically ventilated patients with arhs , low co is multifactorial and could be due to rv systolic dysfunction , tricuspid regurgitation , ventricular interdependence ( dilatation of the rv shifting the interventricular septum toward the left and decreasing the lv distensibility and preload ) , arrhythmias or suboptimal preload . rv diastolic dysfunction causes impaired rv filling and high diastolic rv and right atrial ( ra ) pressures leading to organ congestion . the causes and precipitating events of arhs are summarized in table 2 . precipitating events / causes of arhs in the icu ( 7 - 15 ) . arhs = acute right heart syndrome ; icu = intensive care unit ; rv = right ventricular ; lv = left ventricular . arhs in acute respiratory distress syndrome ( ards ) ards is one of the most common causes of arhs secondary to rv pressure overload ( acute cor pulmonale ) . in critically ill ventilated patients with ards , arhs occurs in 61% of patients submitted to conventional tidal volume mechanical ventilation ( mv ) and 25% of those receiving lung protective mv using low tidal volumes [ 15 , 16 ] . apart from mv , the pathologic features of the syndrome per se , contribute to increased pulmonary vascular tone , acute pulmonary arterial hypertension ( pah ) and cor pulmonale . contributors to elevated pulmonary vascular resistance ( pvr ) in ards include : vasoconstrictor : vasodilator imbalance ( excess et-1 , 5ht , pde , reduced no and prostanoids ) , endothelial injury , hypoxic pulmonary vasoconstriction ( 80% arteriolar ) , hypercapnia ( including permissive hypercapnia ) , acidemia , in situ thrombosis and pulmonary vascular remodelling ( muscularization of non - muscularized arteries ) [ 8 , 16 , 17 ] . arhs in the setting of massive pulmonary embolism ( pe ) critically ill patients are at risk of pe despite thromboprophylaxis [ 3 , 18 ] . in a ten - year retrospective study , vieillard - baron et al . showed that arhs was present in 61% of medical intensive care unit ( icu ) patients with massive pe and carried a 23% mortality . the normal rv can generate a mean pulmonary artery pressure up to 40 mmhg , requiring 50 - 75% of the pulmonary vasculature to be occluded by emboli before acute rv failure occurs . hypoxemia induced by the emboli results in pulmonary vasoconstriction and the physiological response to platelet activation leading to release of vasoactive agents such as serotonin , thromboxane and histamine , causes further increase in pvr and rv pressure overload [ 19 , 20 ] . in severe sepsis and septic shock rv systolic dysfunction in sepsis is directly associated with markers of endothelial dysfunction ( endothelin 1 , vascular cellular adhesion molecule 1 ) and directly related to the severity of sepsis . a proposed mechanism for arhs in sepsis is increased pvr secondary to sepsis - induced endothelial cell injury and altered vaso - reactivity , despite concomitant decrease in systemic vascular resistance ( svr ) . substantial increases in pvr also occur when the left ventricle needs to considerably increase the cardiac output in order to compensate for the fall in the svr , causing further increase in rv afterload [ 21 , 22 ] . diagnosis of arhs in the critically ill clinical features the clinical features of arhs , including acute onset shortness of breath , orthopnea and bilateral lower extremity edema , are non - specific and difficult to identify in the sedated critically ill patient . increased oxygen requirements or sudden cardiovascular collapse might be the chief clinical manifestations of arhs in a mechanically ventilated patient . other prominent clinical signs include atrial or ventricular arrhythmias , raised jugular venous pressure and gallop rhythm at the left sternal edge , systolic murmur of tricuspid regurgitation , organomegaly , signs of deep venous thrombosis ( in the context of venous thromboembolism ) . it is important to consider arhs in persistent respiratory weaning failure ( rv dysfunction leads to an imbalance between ventilator needs and cardiorespiratory capacity ) , especially in patients with lv systolic dysfunction [ 6 , 9 , 24 , 25 ] . a high index of suspicion is needed in high risk patients such as those with pre - existing pah and recent deep venous thrombosis . available bedside studies include : chest x - ray ( cxr ) , electrocardiography ( ekg ) , arterial blood gas ( abg ) analysis , hemodynamic and echocardiographic diagnostic tools . however , cxr can not be utilized to confirm the diagnosis of arhs and should only contribute to the diagnostic approach by ruling out conditions that mimic arhs in the icu , such as atelectasis , pleural effusions , pulmonary edema and pneumothorax . showed that qr in v1 is a strong predictor of rv dysfunction , and it is highly associated with troponin leakage and myocardial shear stress . it has also been demonstrated that in patients with right bundle branch block , r duration in lead v1>100 ms is predictive of rv systolic dysfunction . other ekg findings suggestive of rv strain include inversion of t waves in leads v1-v4 and the classic s1q3t3 pattern . acute anterior q - wave pattern in leads v1-v3 , as well as a right - sided q pattern in leads v3r - v6r , might suggest rv infarction . arterial blood gas analysis abg analysis may reveal grossly impaired gas exchange and low cardiac output might result in acidemia with lactic acidosis due to tissue hypoperfusion . hemodynamic bedside diagnostic modalities central venous catheters and central venous pressure an accurately placed central venous catheter ( in the superior vena cava ) , can provide information on cvp and used as a surrogate for rv end - diastolic volume ( rvedv ) and rv end - diastolic pressure ( rvedp ) [ 25 , 28 ] . in severe tricuspid regurgitation causing arhs , a broad , tall systolic c - v wave is seen due to abnormal systolic filling of the right atrium ( ra ) and the cvp trace is said to be ventricularized because it resembles right ventricular pressure [ 25 , 28 ] . rvedp reflects rvedv ( which is proportional to preload ) only when ventricular compliance is normal . therefore , in conditions such as pah , tamponade and myocardial ischemia , where rv compliance is decreased , cvp is likely to be raised and can not be accurately assessed [ 28 , 29 ] . right heart catheterization right heart catheterization using a pulmonary artery catheter ( pac ) is frequently required when arhs is clinically suspected and interpretation of imaging studies is difficult or inconclusive . hemodynamic data obtained from an accurately placed pac , by thermodilution , may provide diagnostic clues and guide therapy . pac allows direct simultaneous measurement of ra , rv , pa and pulmonary artery wedge pressures and indirect measurement of cardiac output , cardiac index ( ci ) , rv stroke work index , mixed venous oxygen saturation , pvr and svr [ 29 , 30 ] . hemodynamically , arhs is suspected if ra pressure > 8 - 10 mmhg , or ra pressure to pulmonary capillary wedge pressure 0.8 ( isolated rv failure ) and ci is low . in the presence of rv - pa gradient > 25 mmhg , rv outflow tract obstruction should be excluded by echocardiography . in the context of pah and suspected arhs , right heart catheterization allows assessment of left - sided heart disease and its contribution to pah . besides , calculation of pvr and svr help decide whether pulmonary or systemic vasodilators / pressors are needed and monitor response to therapy . in patients with pre - existing pah , a decrease in pa pressure might reflect low rv ejection fraction and worsening rv dysfunction . arterial pulse contour analysis arterial pulse contour analysis enables calculation of co , pulse pressure variation ( ppv ) , stroke volume variation ( svv ) and svr , from the arterial pulse pressure waveform , in mechanically ventilated patients . dynamic indices ( svv , ppv ) have been used to predict preload responsiveness and monitor the hemodynamic effect of volume expansion in critically ill patients . wyler von ballmoos et al . reported that ppv is not accurate predictor of fluid responsiveness in mechanically ventilated patients with acute pah ( at risk of arhs ) , early after cardiac surgery and in septic shock . in the context of a pressure overloaded rv , increased ppv values are related to an increase in the rv afterload and not to a decrease in rv preload and therefore , further volume expansion could potentially be harmful [ 33 , 34 ] . however , it could be reasonably contended that lack of response to a fluid challenge , while ppv or svv is high , could be seen as an indicator of rv dysfunction necessitating further investigations . bedside imaging modalities transthoracic ( tte ) and transesophageal echocardiography ( tee ) are bedside diagnostic tools which also provide rapid risk stratification and could potentially direct therapeutic strategies . in experienced hands , echocardiography allows assessment of the rv performance and loading conditions . useful echocardiography - derived measures of rv function , when arhs is clinically suspected are outlined in table 3 . echocardiographic quantitative parameters pointing towards arhs ( 36 - 38 ) rv = right ventricular ; tapse = tricuspid annular plane systolic excursion ; 2drvfac = two - dimensional right ventricular fractional area change ; rimp = right ventricular index of myocardial performance ; e / a = early ( e ) to late ( a ) ventricular filling velocities ratio ; rvot = right ventricular outflow tract . tte is an easy and non - invasive way to assess the size and kinetics of the rv . the diagnosis of arhs due to rv pressure overload , with tte , has good positive predictive value for indirect diagnosis of massive pe . main limitations of tte in critically ill patients ventilated with high level of positive end - expiratory pressure ( peep ) include : inadequate imaging due to interposition of the inflated lung between the heart and the chest wall , low diagnostic accuracy in the patients with pre - existing cardiopulmonary disease , the operator dependent nature of tte . it has been suggested that in the presence of significant and otherwise unexplained rv strain without clots present on tte , tee should rapidly follow at the bedside , providing there is local availability and expertise . tee is a semi - invasive procedure and commonly reported complications associated with tee in critically ill patients receiving mv , include : hypo- or hypertension , dysrrhythmias , trauma to the gastrointestinal tract , hypoxemia and dislodgment of endotracheal or nasogastric tubes . the over - all complication rate associated with tee use is low and it is estimated to be approximately 2.6% . additional imaging modalities computed tomography ( ct ) ct pulmonary angiography ( ctpa ) is being used increasingly as a diagnostic tool in pe , with documented sensitivities of 50 - 100% and specificities of 81 - 100% . ctpa has become the preferred diagnostic modality for suspected arhs due to pe , in hemodynamically stable icu patients . chest ct signs suggestive of arhs include : flattening or displacement of the intraventricular septum toward the lv , reflux of contrast into the inferior vena cava , rv diameter ( rvd ) to lv diameter ( lvd ) ratio on axial sections greater than 1.0 ( rvdaxial / lvdaxial > 1 ) . cardiovascular magnetic resonance ( cmr ) cmr is the most sensitive method to assess the rv size and function . however , cmr is rarely used for icu patients receiving mv , as the mr environment carries significant risks to patients during transportation and prolonged periods in the mr scanner . the usefulness of laboratory tests such as d - dimmer , troponins and b - type natriuretic peptide levels , as diagnostic tests in icu patients with suspected rv failure , is limited , as they are non - specific and confounded in the context of critical illness [ 41 , 42 ] . in summary , in critically ill patients with clinically suspected arhs , echocardiography ( tte and/or tee ) and right heart catheterization are the preferred diagnostic modalities . if pe is the most likely cause of arhs , then ctpa is necessary to confirm the diagnosis , provided the patient is suitable for transfer to radiology . in any given scenario , the diagnostic approach will depend upon the expertise and availability of the different diagnostic modalities . the principles and key components of arhs management include reversal of precipitating events and control of contributing factors ( hypoxemia , hypercapnia , anemia , acidemia , sepsis , dysrrhythmias ) , fluid volume optimization , maintenance of perfusion pressure , positive inotropy , use of pulmonary vasodilators and protective mv [ 12 , 43 ] . control of contributing factors , general icu care and reversible causes of arhs infection prevention , treatment of sepsis in accordance with the surviving sepsis campaign bundles , normoxia , normocapnia , thromboprophylaxis , peptic ulcer prophylaxis , correction of acid - base imbalance and electrolytes and glycemic control are mandatory and applied to most icu patients . pulmonary vasodilators and/or inodilators ( in acutely decompensated pah ) , thrombolysis ( in massive pe ) , revascularization ( in rv infarction ) and sequential av pacing and/or cardioversion ( in significant dysrrhythmias ) , could potentially correct the abnormal rv physiology . rv ejection fraction is dependent on rv pre - load , in the abscence of pah and it is likely that rv output will be inadequate in hypovolemia . the rv can increase the stroke work through an increase in rv free wall stretch ( via the frank - starling mechanism ) . a systematic review of 24 studies demonstrated a poor relationship between cvp and intravascular fluid status and the inability of cvp / delta - cvp to predict the hemodynamic response to a fluid challenge [ 32 , 45 ] . depending on where the patient is on the frank - starling curve , some may be adequately resuscitated with a cvp of 6 - 7 mm hg , while others may still be intravascularly volume depleted at a cvp of 10 mm hg . in a recent meta - analysis , marik et al . showed that there is paucity of data to support the widespread practice of using cvp to assess intravascular fluid status and guide fluid therapy . more reliable hemodynamic assessment tools , such as pac , pulse contour analysis , tte and/or tee when available , may be utilized to guide fluid therapy in arhs . showed that in critically ill medical patients with circulatory failure ( defined by ci 2 ) , due to massive pe , fluid loading with 500 ml of colloid increased the cardiac index significantly and improved hemodynamic status . if the hemodynamic response to initial fluid challenge is poor , in the context of arhs , further volume loading may cause rv overdistension , increased ventricular interdependence , decreased lv filling and rv ischemia , leading to worsening shock . in rv volume overload , acute kidney injury due to venous congestion ( cardiorenal syndrome ) , continuous veno - venous hemofiltration ( cvvh ) facilitates greater clinical improvement compared with aggressive diuretic therapy , in heart failure patients , who are diuretic resistant . the role of vasopressors in arhs in order to preserve adequate right coronary blood flow , systemic pressure should be maintained above the pa pressures . it has been shown that in patients with sepsis , pah and rv dysfunction , norepinephrine increases systemic pressure through alpha-1 receptor agonism and may improve the rv oxygen supply / demand ratio , but this potentially beneficial effect on rv ejection fraction may be offset by a concomitant increase in pvr and rv afterload , at high doses ( > 0.5 mcg / kg / min ) [ 43 , 49 ] . besides , norepinephrine , through beta-1 receptor agonism could potentially improve rv - pa coupling and co . low dose vasopressin ( 0.033 - 0.067 u / min ) mediates pulmonary arterial vasodilation and may be useful in vasodilatory shock and pulmonary vascular dysfunction , especially in norepinephrine resistant patients [ 43 , 49 , 50 ] . inotropes and inodilators in arhs dobutamine ( beta-1 receptor agonist ) can be used as the first - line inotropic agent in arhs due to rv contractile dysfunction . low dose dobutamine ( 2 - 5 mcg / kg / min ) increases ci , sv and decreases pvr and svr [ 12 , 43 , 51 ] . at higher doses ( > 10 mcg / kg / min ) dobutamine causes tachycardia , increased oxygen consumption , increased pvr and leads to systemic hypotension and addition of a vasopressor might be required [ 12 , 51 ] . high quality evidence suggests that dopamine is associated with increased tachyarrhythmias and is not recommended in cardiogenic shock . it has been demonstrated that in patients with septic shock and arhs , who are unresponsive to fluid loading , dopamine or dobutamine , epinephrine improves rv contractility in spite of a rise in mean pap by 11% ( p<0.05 ) . selective phosphodiresterase ( pde ) iii inhibitors ( enoximone , milrinone , amrinone ) , augment myocardial contractility and cause systemic and pulmonary vasodilaton , by increasing cyclic adenosine monophosphate ( camp ) and thus reducing pa pressures and improving rv function in patients with arhs due to pressure overloaded rv . it has been demonstrated that levosimendan , a calcium sensitizer with pulmonary vasodilator properties ( inodilator ) , improves rv performance in arhs secondary to sepsis - induced ards and in experimental arhs restores rv - pa coupling better than dobutamine . in arhs , levosimendan has been shown to reduce the increased rv afterload and ventricular interdependence , improve rv contractility and rv diastolic function , without significant increase in oxygen consumption , mediated by opening of sarcolemmal and mitochondrial potassium - adenosine triphosphate channels [ 54 , 55 ] . although levosimendan is indicated for the treatment of acute heart failure ( class of recommendation iia , level of evidence b ) , it is has not yet been approved in all countries [ 54 , 55 ] . pulmonary vasodilation in arhs the goals of pulmonary vasodilation in arhs are : 1 ) decrease pvr and impedance ; 2 ) increase rv stroke volume and output ; 3 ) avoid systemic hypotension and maintain coronary perfusion ; 4 ) avoid hypoxemia from ventilation - perfusion mismatch . it is recommended that inhaled nitric oxide ( no ) , which increases intra - cellular cyclic guanosine monophosphate ( cgmp ) , should be considered as short term therapy to improve pao2/fio2 ratio and co , in ventilated patients with arhs secondary to ards . it has also been suggested that no may be effective in stabilizing patients with arhs due to massive pe until more definitive treatment is available [ 29 , 56 ] . prostanoid formulations ( epoprostenol , iloprost ) are potent pulmonary and systemic vasodilators with anti - thrombotic and anti - proliferative actions . they reduce pvr and improve rv function and they have been used in arhs due to rv pressure overload . it has been shown that use of intravenous epoprostenol in mechanically ventilated patients with ards reduces pvr and improves rv performance . sildenafil , a pde 5 inhibitor , increases downstream cgmp signaling and potentiates the beneficial effects of no . showed that mechanically ventilated patients with arhs from pah , who were dependent on dobutamine , were treated with oral sildenafil and in many cases they were successfully weaned from inotropic and ventilatory support . mechanical ventilation strategies in arhs optimal mv ventilation management in arhs consists of : avoidance of hypoxemia , hypercapnia , high levels of peep ( > 10 cmh2o ) and both high and low extremes of lung volumes and use lung protective ventilation strategies if possible [ 43 , 60,61,62 ] . the rv afterload is governed by pvr which is directly affected by changes in lung volume . this is due to the elastic recoil forces of the lung parenchyma leading to extra - alveolar vessel collapse and terminal airway collapse leading to alveolar hypoxia and hypoxic pulmonary vasoconstriction ( hpv ) and at high lung volumes due to collapse of the alveolar vessels via stretch of the alveolar wall . when pvr is plotted against lung volume , a typical u - shaped curve occurs with the lowest pvr occurring at functional residual capacity ( frc ) ( figure 2 ) . effect of changing lung volume on pulmonary vascular resistance ( pvr ) ( 62 ) . rv = residual volume ; frc = functional residual capacity ; tlc = total lung capacity . schmitt et al . assessed the impact of peep on the rv outflow impedance using doppler data obtained by tee , in mechanically ventilated icu patients with ards . they demonstrated that high peep ( 134 cmh2o ) was associated with increased rv afterload and worsening rv systolic dysfunction . the significant decrease in the incidence of arhs ( from 61% to 25% ) in ards since ardsnet trial was published , reflects a change in mv practice and suggests that lung protective strategies ( tidal volume : 6 - 8 ml / kg predicted body weight ( pbw ) , low plateau pressures and peep ) reduce the incidence of arhs . in patients with arhs , during lung protective ventilation , permissive hypercapnia should be avoided as acute hypercapnia could lead to pulmonary vasoconstriction or exacerbate hypoxic pulmonary vasoconstriction and could potentially worsen rv dysfunction . a prospective observational study , which evaluated the relative roles of acute permissive hypercapnia and peep variations on rv function , in severe ards patients , showed that increasing peep at constant pplat induces acute hypercapnia that may impair rv function and decrease ci . it is therefore recommended that in cases of arhs , lung protective ventilation should be gradually adapted to limit hypercapnia and rv overload . in mechanically ventilated icu patients with arhs , refractory hypoxemia and/or hypercapnia and high peep requirements , extracorporeal membrane oxygenation ( ecmo ) could be used as a bridge to the recovery of respiratory function . oxygenation and carbon dioxide clearance are provided by the extracorporeal circuit , minimizing pulmonary vasoconstriction due to hypoxemia and/or hypercapnia [ 66 , 67 ] . in patients with arhs due to severe ards , where lung protective ventilation may not be adequate in managing hypercapnic acidosis , extracorporeal carbon dioxide ( ecco2 ) removal devices are an option , as they are less invasive than ecmo and may play a role in instituting ultra - protective lung ventilation ( tidal volume : 4 ml / kg pbw ) . it should be noted that the cardiac consequences of weaning from mv may be responsible for weaning failure in patients with arhs . in these patients , an increase in weaning - induced rv afterload may occur due to marked increase in work of breathing , hypoxemia or high intrinsic peep , leading to further worsening rv enlargement during weaning . this may result in leftward shift of the interventricular septum , impeding lv diastolic filling and lv output ( ventricular interdependence ) , causing pulmonary edema and failure to wean from mv . mechanical circulatory support low cardiac output syndrome caused by arhs after cardiac surgery , particularly coronary artery bypass graft surgery and heart transplant , may be an indication for intra - aortic balloon pump ( iabp ) [ 70 , 71 ] . it has been demonstrated that iabp improves hemodynamics and rv efficiency in acute ischemic rv failure . however , a recent rct failed to demonstrate any mortality benefit in patients with cardiogenic shock complicating acute myocardial infarction . veno - arterial ( va ) ecmo has been used as a salvage therapy in cases of arhs due to massive pe and refractory cardiogenic shock , after systemic thrombolysis . it can be used as a means of unloading the rv and supporting systemic circulation , in medically refractory rv failure with accompanying hypotension and end - organ failure and as a bridge to transplant [ 74 , 75 ] . right ventricular assist devices ( rvads ) in arhs may be used as a bridge to recovery or transplant , or as a definitive surgical treatment , in primary rv dysfunction . in patients who are successfully weaned from the rvad , it has been suggested that rvads should be avoided in patients with arhs secondary to rv afterload resistance ( with severely elevated pvr ) , as pumping blood into the pa could potentially cause worsening pah and lung injury , whereas co and ci remain low . in such cases the use of mechanical cardiovascular support devices depends largely on local availability of specialized facilities , cardiopulmonary pathophysiology expertise and operator experience . arhs is difficult to diagnose in the critically ill as those patients have ongoing physiological derangement presenting the intensive care specialists with a diagnostic dilemma . cardiac echo and right heart catheterization are invaluable diagnostic tools in the assessment of the rv at the bedside , which also provide a rapid risk stratification and could direct treatment strategies . characterizing , identifying and correcting reversible factors is of paramount importance . minimizing rv afterload ( pulmonary vasodilators , inodilators , rv protective mv strategies ) and maximizing rv performance ( preload , inotropy , mechanical circulatory support ) are the major components of arhs management . need for mechanical circulatory support , merits referral to specialized treatment centres , if there is insufficient local expertise or capacity . there is lack of definitive data regarding the management of arhs in icu patients , without pre - existing cardiopulmonary disease . well - designed and adequately powered rcts are required to estimate the prevalence of arhs among critically patients receiving mv , improve the understanding of its mechanisms in the context of critical illness and evaluate the efficacy of therapy guided by invasive and non - invasive hemodynamic monitoring tools .

introduction : diabetes is a group of metabolic diseases characterized by hyperglycemia , and represents a disease of the modern age , disease of the 21st century . prevention of this disease is listed as imperative . aim of this article was to evaluate questionnaires on the assessment of risk factors for diabetes mellitus type 2.material and methods : a total of 540 questionnaires handed out randomly to citizens of canton sarajevo of all ages , sexes and educational levels ( in january 2016 ) were analyzed.results:analyzed questionnaires showed relatively low risk of getting diabetes in the next ten years in the majority of the population . these results are rather encouraging but may in some way be in confrontation with the statistics which show a rapid outburst of diabetes.conclusion:the life - style is the main reason for such a thing to happen , and looking at these questionnaires , we might get the feeling that we really do live in a , conditionally speaking , physically active society . that , from our everyday experience is not entirely true . it would be wise to continue doing research on this topic on the territory of bosnia and herzegovina . acute right heart syndrome is a sudden deterioration in right ventricular performance , resulting in right ventricular failure and confers significant in - hospital morbidity and mortality . in critically ill patients , the syndrome is often undiagnosed and untreated , as these patients do not usually exhibit the common clinical manifestations of the condition , making the diagnosis challenging for the intensivist . in this narrative review we focus on the pathophysiology of acute right heart syndrome , in critical illness , diagnostic modalities used to assess right ventricular function and management of acute right heart syndrome , including mechanical ventilation strategies and circulatory support .

in rare conditions , bacteria from carbuncles can spread into the bloodstream and migrate to other areas of the body , causing serious complications such as septicemia and infections in the liver , bones , joints , heart , and central nervous system . we describe an original case of forehead carbuncle with intractable headache , later confirmed as a subgaleal abscess . a 74-year - old female visited our hospital due to a 2 cm , painful , erythematous nodule with a small necrotic plug on her forehead , which had been resident for 2 days . she had suffered for 5 years from diabetes mellitus , which was not aggressively controlled . one day later , the patient visited our emergency room again due to progressive erythema and worsening headache . she had mild fever ( 37.7c ) and bilateral periorbital soft tissue swelling with local heat . laboratory data showed elevated white blood cell count ( 10.521,000 cells/l ) and c - reactive protein levels ( 78.90 preliminary diagnosis suspected cellulitis ; she was admitted to kaohsiung municipal ta - tung hospital , kaohsiung , for further management . however , her headache kept worsening and interfered with her sleep over the next 2 days . she described the headache as severe , persistent aching all over her head , which was not associated with postural change . nonsteroidal anti - inflammatory drugs and tramadol hydrochloride were administered but completely in vain . in the fear of central nervous system involvement however , brain computed tomography ( ct ) was still arranged due to intractable headache . brain ct showed no intracranial lesion , but subgaleal emphysema and abscess were suspected ( figure 1 ) . a neurosurgeon was then consulted , and operative debridement was promptly arranged . during the operation , widespread subgaleal abscess was noted and drained . as previously described in the case history section , this 74-year - old female had intractable headache , which developed in close relationship with subgaleal abscess . in addition , her headache resolved within 3 months after successful treatment of subgaleal abscess with operative debridement and antibiotics . according to the international classification of headache disorders ( ichd-2 ) criteria , headache disorders attributed to extracranial infection of the head ( such as ear , eye , and sinus infection ) are coded as subtypes 11 . headache or facial pain can be attributed to a disorder of the cranium , neck , eyes , ears , nose , sinuses , teeth , mouth , or other facial or cranial structures.1 carbuncle is a common dermatologic disease , and staphylococcus aureus is the pathogen responsible in most cases . though some authors advocate that systemic antimicrobial treatment is not needed for simple furuncles and carbuncles,2 we agree that incision and drainage with ancillary antimicrobial therapy is recommended for patients with immunosuppression or comorbidities , extremes of age ; rapid progression to cellulitis and the lack of an adequate response to incision and drainage are also indications for such treatment.3 however , empiric antibiotics for carbuncle , even vancomycin , did not work for the unusual bacterial culture of klebsiella in this patient . although klebsiella is strongly associated with infections in patients with diabetes , skin and soft tissue infections from klebsiella are still uncommon.4 this case reminds us that empiric , systemic antibiotics for carbuncles and cellulitis may respond poorly in situations as described in the case history section . the most common cause of subgaleal abscess is direct inoculation of microbes into the subgaleal space following scalp trauma . however , subgaleal abscess may result from hematogenous infection or contiguous spread , and the diagnosis may not be initially obvious.5 carbuncles may progress to cellulitis and cause redness of the skin , swelling , and pain . however , carbuncles located on the middle of the face may raise concern , especially when the patient is elderly and immune - compromised . intractable headache that is unresponsive to the standard medications and therapies utilized in the treatment of headaches also indicates the need for further survey . head ct is often needed in the diagnosis of subgaleal abscess , and if operative debridement is not promptly performed , subgaleal abscess may further progress to life - threatening septicemia , osteomyelitis , and even subdural or brain abscess or meningitis.6 there are no focal neurological signs or specific symptoms in the early stages of subgaleal abscess . thus , when encountering patients with intractable headaches , unusual causes should be kept in mind , which may include moyamoya syndrome,7 headache after botulinum a exotoxin injections,8 acquired immunodeficiency syndrome - related lymphoma confined to bone,9 migraine in obese individuals,10,11 and children with both migraine and periodic limb movement disorders in sleep.12 in conclusion , this case highlights that special attention should be paid to elderly and immune - compromised patients with carbuncles located on the middle of the face , especially when accompanied by intractable headache . hydroxyl radicals , generated by cellular oxidative stress and inflammation , damage nucleobases or deoxyribose sugars , or both in dna . at 2-deoxyguanosines , hydroxyl radical - mediated hydrogen abstraction at the deoxyribose c5-position followed by attack at the guanine c8 carbon forms an n7-centered radical , which may be oxidized to diastereomeric 8,5-cyclo-2-deoxyguanosines ( cdg ) . the 8,5-cyclo-2-deoxyadenosines ( cda ) have also been characterized . these 8,5-cyclopurine-2-deoxynucleosides have been detected at the nucleotide level , in dna , and cells in vitro , in human urine , and in vivo . they might contribute to neurologic disease in xeroderma pigmentosum complementation group c ( xp - c ) patients . they are also believed to play roles in cockayne syndrome , breast and ovarian cancer , and familial mediterranean fever . it has been reported that s - cdg does not block primer elongation by klenow dna polymerases , and datp is preferentially incorporated opposite the lesion in vitro . however , in escherichia coli , s - cdg blocks dna replication and is refractory to repair . most are s - cdg a transitions , although s - cdg t transversions and low levels of deletions of the 5-neighbor dc are also observed . for both cdg and cda , the diastereomeric ratio at the c5-position depends on experimental conditions and dna conformation . computational studies predicted that the incorporation of the cda stereoisomers into dna would result in helical distortions at the lesion site . both the r- and the s - diastereomers of the 8,5-ca ribonucleoside have been crystallized , and both exhibit the anti conformation about the n - glycosidic bond with o4c1n9c8 = 30 or 27 , respectively . the fused six - member ring c8n9c1o4c4c5 adopts the half - chair conformation with the o4 and c4 out of plane . the ribose adopts the o4-exo ( 0 t ) pseudorotation with p = 289 and m = 48. molecular mechanics calculations predicted that the cda diasetereomers maintain the o4-exo pseudorotation when placed opposite dt in dna . the nmr data and ab initio calculations suggest that incorporation of the s - cda into di- or trinucleotides does not change the o4-exo pseudorotation . recently , we reported the structure of the s - cdgdc pair in 5-d(gtgcxtgtttgt)-35-d(acaaacacgcac)-3 , containing the dna sequence of p53 codons 272275 ( x = s - cdg , scheme 1 ) . however , the s - cdg deoxyribose shifts to the o4-exo pseudorotation with p = 280. this altered backbone torsion angles from 50 to 67 and from 120 to 149 , as compared with canonical b - dna . additionally , the torsion angles and are changed from 180 to 87 and from 120 to 157 , respectively . the twist and base pair shift helicoidal parameters are perturbed at the cg and xc base pairs . the purine ring is anti about the n - glycosidic bond , and the fused six - membered ring adopts the half - chair conformation with o4 and c4 out of plane . here , we report the structures of the duplexes 5-d(gtgcxtgtttgt)-35-d(acaaacaygcac)-3 ( x denotes s - cdg ; y denotes either da or dt , scheme 1 ) . these model the situation following mis - incorporation of dttp opposite s - cdg , leading to s - cdg a transitions , or following mis - incorporation of datp opposite s - cdg , leading to s - cdg t transversions in e. coli . the s - cdgdt mismatch pair adopts a wobble base pairing , providing a plausible rationale for the s - cdg a transitions . for the s - cdgda mismatch pair , both s - cdg and da intercalate , but no hydrogen bonding is observed between s - cdg and da . this is consistent with the lower levels of s - cdg t transitions in e. coli . the oligodeoxynucleotide 5-d(gtgcxtgtttgt)-3 ( x = s - cdg ) was synthesized and characterized as reported.the oligodeoxynucleotides 5-d(gtgcgtgtttgt)-3 and 5-d(acaaacaygcac)-3 ( y = da or dt ) were synthesized and purified by anion - exchange chromatography ( midland certified reagent co. , midland , tx ) . the oligodeoxynucleotides 5-d(gtgcgtgtttgt)-3 or 5-d(gtgcxtgtttgt)-3 were annealed at 1:1 stoichiometry with the complementary oligodeoxynucleotide 5-d(acaaacaygcac)-3 in 10 mm nah2po4 , 100 mm nacl , and 50 m na2edta ( ph 7.0 ) . the solutions were heated to 95 c for 10 min and cooled to room temperature . the duplexes were isolated using dna grade hydroxylapatite with a gradient from 10 to 200 mm nah2po4 in 100 mm nacl and 50 m na2edta ( ph 7.0 ) and desalted using sephadex g-25 . melting temperatures of the dna duplexes were measured by uv / vis spectroscopy at 260 nm in 10 mm nah2po4 , 100 mm nacl , and 50 m na2edta ( ph 7.0 ) . the thermal scans proceeded from 10 to 80 c with an interval of 1 c . samples for the nonexchangeable protons were dissolved in 500 l in 10 mm nah2po4 , 100 mm nacl , and 50 m na2edta ( ph 7.0 ) . they were exchanged with d2o and suspended in 280 l of 99.996% d2o , and the ph was adjusted with dilute dcl or naod . magnitude correlated spectroscopy ( cosy ) spectra were recorded with 512 real data in the t1 dimension and 2048 real data in the t2 dimension . total correlation spectroscopy ( tocsy ) spectra were recorded with a mixing time of 80 ms . the exclusive correlation spectroscopy ( e - cosy ) spectra were recorded with 1024 real points in the t1 dimension and 4096 real points in the t2 dimension . the spectra were zero - filled during processing to create a matrix of 2048 16384 points . nuclear overhauser effect spectroscopy ( noesy ) spectra were recorded with 512 real points in the t1 dimension and 2048 real points in the t2 dimension . noesy spectra were zero - filled during processing to create a matrix of 1024 1024 real points . noesy experiments used time - proportional phase increment ( tppi ) quadrature detection and mixing times of 60 , 150 , 200 , and 250 ms . the relaxation delay was 1.5 s. data were processed using the program topspin and analyzed with the program sparky . samples for the observation of exchangeable protons were dissolved in 500 l of 10 mm nah2po4 and 100 mm nacl , 50 m edta ( ph 7.0 ) containing 9:1 h2o : d2o ( v / v ) ( ph 7.0 ) . the watergate sequence was used for water suppression , with a nuclear overhauser enhancement ( noe ) mixing time of 250 ms . the p h experiments were carried out at the h frequency of 600 mhz . h3 j couplings were applied to determine the phosphodiester backbone conformation.p chemical shifts were referenced using indirect shift ratios . integration footprints were defined using noe cross - peaks obtained at a mixing time of 250 ms . noe intensities from data obtained at mixing times of 60 , 150 , 200 , and 250 ms to check for the presence of spin diffusion effects were determined by volume integrations . for each mixing time , these were combined as necessary with intensities generated from complete relaxation matrix analysis of a starting structure to yield a hybrid noe intensity matrix . the program mardigras iteratively refined the hybrid intensity matrix and optimized agreement between calculated and experimental noe intensities . the randmardi algorithm carried out iterations , randomizing peak volumes within limits specified by the input noise level . calculations were initiated using isotropic correlation times of 2 , 3 , and 4 ns . analysis of these data yielded distance restraints used in restrained molecular dynamics ( rmd ) calculations ( table s3 in the supporting information ) and the corresponding standard deviations for the distance restraints . the pseudorotations ( p ) were estimated by examining the jhh of deoxyribose protons . the data were fit to curves relating the coupling constants to p , deoxyribose pucker amplitude ( ) , and the percentage s type conformation . the p and ranges were converted to the dihedral angles 04 . to obtain backbone torsion angle restraints for the modified , flanking , and terminal base pairs , coupling constants measured from h p heternuclear multiple bond correlation spectroscopy ( hmbc ) spectra were applied to the karplus relationship to determine the dihedral angle ( c4c3o3p ) , related to the h3c3o3p angle by a 120 shift . the ( c3o3p o5 ) torsion angles were calculated from the correlation between and in b - dna . at all other base pairs , crick hydrogen - bonding restraints minimized propeller twisting between base pairs , except at the xt base pair in the s - cdgdt mismatched duplex and at the xa and at base pairs in the s - cdgda mismatched duplex . the partial charges for the cdg nucleotide were obtained from density functional theory ( dft ) calculations , utilizing the b3lyp/6 - 31 g * basis set and the program gaussian . the starting structures were generated from a and b type dnas by constructing a bond between g5 c8 and g c5 followed by 200 iterations of potential energy minimization using the conjugate gradients algorithm . the generalized born ( gb ) model with parameters developed by tsui and case was used for implicit water simulation . the program complete relaxation matrix analysis ( corma ) was utilized to calculate the noe intensities from the structures emergent from calculations . the duplex containing the s - cdgdt base pair exhibited a melting temperature ( tm ) of 38 1 c . the unmodified dgdt mismatched duplex exhibited a tm of 43 c under the same conditions ( figure 1a ) . thus , the incorporation of s - cdg reduced the tm by 5 c . the duplex containing the s - cdgda pair exhibited a tm of 31 1 c . the unmodified dgda mismatched duplex exhibited a tm of 39 1 c ( figure 1a ) . h nmr spectra of the duplex containing the s - cdgdt pair were compared with the unmodified mismatched duplex at different temperatures ( figure 1b ) . at the xt base pair , the t n3h resonance was not observed at 5 c . for the modified duplex , the t n3h resonance broadened at lower temperature than did the other thymine imino resonances . h nmr spectra of the duplex containing the s - cdgda base pair at different temperatures are displayed in figure 1c . the imino resonances for base pairs cg , ga , and ta of the unmodified duplex were not observed . moreover , for the unmodified duplex containing the ga mismatch , the c and c h5 h6 scalar couplings were not observed , and that of c was weak . ( a ) uv melting profiles of the mismatched duplexes as compared with the corresponding unmodified duplexes : duplex containing dgda base pair ( ) , duplex containing s - cdgda base pair ( ) , duplex containing dgdt base pair ( ) , and duplex containing s - cdgdt base pair ( ) . ( b ) h nmr of the mismatched duplex containing the s - cdgdt base pair at different temperatures . ( c ) h nmr of the mismatched duplex containing the s - cdgda base pair at different temperatures . the broad resonance at 13.1 ppm observed at 515 c was unassignable the nonexchangeable protons of the s - cdg - modified duplex were assigned based upon the noe sequential connectivity of the base proton h6 or h8 dipolar couplings with h1 deoxyribose protons ( figure 2a , b ) . for the modified strand , the sequential connectivity was observed from g to c. because the s - cdg nucleotide lacked a proton at the c8 carbon , the sequential connectivity exhibited an interruption at x. the x h1 proton was identified at 6.11 ppm ; it exhibited a weak x h1 t h6 noe . the sequential connectivity resumed from t to t. for the modified strand , all of the deoxyribose h1 protons were observed within a narrow chemical shift window , between 5.8 and 6.3 ppm . noe ( 250 ms ) connectivities of base h8/h6 protons with deoxyribose h1 protons of the s - cdg modified duplexes . ( a ) modified strand for the duplex containing the s - cdgdt base pair . ( b ) complementary strand for the duplex containing the s - cdgdt base pair . ( c ) modified strand for the duplex containing the s - cdgda base pair . ( d ) complementary strand for the duplex containing the s - cdgda base pair . the assignments of x deoxyribose protons were made by analysis of scalar and dipolar couplings . figure 3a displays a tile plot derived from a noesy spectrum obtained at 60 ms mixing time . x h1 exhibited dipolar couplings with h2 and h2 ; weak scalar couplings were also observed . h3 exhibited dipolar couplings with h2 , h2 , and h4 , whereas the scalar couplings were not observed . the geminal h2 and h2 protons were not resolved . for the remainder of the duplex , the h2 , h2 , h3 , and h4 deoxyribose resonances were unequivocally assigned . the absolute configurations of the geminal h2 and h2 protons were assigned from their noes to h1 and h3. with the exception of the unresolved resonances for x , g , and t , h2 exhibited a weaker noe with h1 than did h2 , whereas it exhibited a stronger noe with h3 than did h2. the resonance assignments of the nonexchangeable dna protons are tabulated in table s1 in the supporting information . expansions of noesy spectra ( 60 ms ) showing the assignment of s - cdg nonexchangeable protons . ( a ) duplex containing s - cdgdt base pair . ( b ) the resonances of the base imino protons were assigned based on sequential connectivity in noesy spectra , and the assignments were supported by noes to the amino protons of watson the noe sequential connectivity was observed from t g to g and from t g t t t to g. at the 5-neighbor base pair , g n1h exhibited noes with cn h1 and n h2 . at the 3-neighbor base pair , the t n3h resonance exhibited noes with a h2 and an h1 . with the exception of the terminal base pairs , the remaining noes arising from watson it exhibited an noe with xnh , which also had an noe with t n3h ( figure 4b ) . ( a ) duplex containing the s - cdgdt base pair ; noe cross - peaks are assigned as follows : a , a h2 g n1h ; b , cnh2 g n1h ; c , cnh1 g n1h ; d , an h1 t n3h ; and e , a h2 t n3h . ( b ) assignment of x n1h in the duplex containing the s - cdgdt base pair ; noe cross - peaks are assigned as follows : f , t n3h xnh ; and g , x n1h xnh . ( c ) duplex containing the s - cdgda base pair ; noe cross - peaks are assigned as follows : h , a h2 g n1h ; i , cnh2 g n1h ; and j , cnh1 g n1h . the scalar couplings of the 2-deoxyribose h1 protons with the h2 and h2 protons were measured from an e - cosy spectrum ( figure s1 in the supporting information ) . the jh1-h2 and jh1-h2 values for x were 2.1 and 4.9 hz , respectively . for t , jh1-h2 and jh1-h2 were 5.3 and 8.8 hz , respectively . with the exception of the terminal nucleotides , the jh1-h2 for other nucleotides were 810 hz , and the jh1-h2 were 57 hz . the j coupling constants for the deoxyribose protons are tabulated in table s2 in the supporting information . the p nmr spectrum of the s - cdg containing duplex was compared with the unmodified mismatched duplex ( figure s2 in the supporting information ) . at the s - cdg nucleotide , the p resonance shifted upfield . the other p resonances were clustered within a modest chemical shift range , centered in the spectral region characteristic of b - dna . chemical shifts of the nonexchangeable protons between the s - cdg - containing duplex andthe unmodified mismatched duplex were compared ( figure 5 ) . remarkable changes were observed at x and the 5- and 3-neighboring nucleotides of the modified strand . c h2 shifted downfield by 0.72 ppm ; x h2 and h2 shifted upfield by 0.21 and 0.51 ppm , respectively ; and t h6 , h1 , and h2 shifted downfield by 0.32 , 0.24 , and 0.23 ppm , respectively . the chemical shift perturbations for the complementary strand were small , with the exception of a h8 and t h1 , which shifted upfield by 0.24 and 0.31 ppm , respectively . chemical shift perturbations of the duplex containing the s - cdgdt base pair . a total of 406 distance restraints , including 263 intranucleotide and 143 internucleotide restraints , were calculated from the intensities of noe cross - peaks ( table s3 in the supporting information ) . a total of 21 noes involving the s - cdg protons were used as restraints . crick base pairing were used , as were 160 empirical torsion angle restraints that were applied to the nonterminal nucleotides . these were justified based upon nmr data , which suggested that structural perturbations were localized at and adjacent to the lesion site . weak wobble base pair restraints were used for the xt base pair , and no torsion angle restraints were used for the cg , xt , and ta base pairs . r1 = |(a0)i1/6 ( ac)i1/6|/|(a0)i1/6| , where a0 and ac are the intensities of observed ( nonzero ) and calculated noe cross - peaks , respectively . the rmd calculations for the s - cdg - containing duplex were performed from a and b form starting structures . ten emergent structures , five each for a- and b - dna starting structures , were obtained and minimized with respect to potential energy . the accuracies of the emergent structures were evaluated by comparison of theoretical noe intensities for the refined structure calculated by the program corma to the experimental noe intensities , to yield sixth root residuals ( r1 ) . these , as well as the residuals for intra- or internucleotide noes , were consistently < 0.1 ( table 1 ) . r1 values for each nucleotide were < 0.15 ( figure s3 in the supporting information ) . thus , the refined structures provided accurate depictions of the noe data . the xt pair adopted the wobble conformation ( figure 6 ) . the xt pair exhibited a shift of 0.8 , displacing c toward the major groove . this pair exhibited a greater than normal opening of 16.8. typical b - dna pairing and stacking interactions were maintained for the remaining base pairs ( table s4 in the supporting information ) . the s - cdg deoxyribose was in the o4-exo , west pseudorotation ( figure 8a ) , with p = 280 and m = 47. the heavy atoms n9 , o3 , and c5 were axial about the deoxyribose ring . pseudorotation , with p = 65 and m = 37. consequently , x h2 was farther from the x purine ring as compared to the h2 protons in b - dna , and c4 h2 was proximate to the x purine ring . with the exception of the terminal nucleotides , the six - membered ring c8n9c1o4c4c5 adopted the envelope ( half boat ) conformation . helicoidal analysis of the backbone torsion angles ( figure s4 in the supporting information ) showed that for s - cdg , the angle shifted from 180 to 78. the angle shifted from 50 to 57. perturbations of the and torsion angles from 120 to 147 and from 90 to 58 , respectively , were also observed . there was also a change for the n - glycosidic torsion angle from 120 to 162. for the complementary t , a perturbation of the torsion angle from 120 to 83 was observed . expanded views of the refined structure of the s - cdg containing duplexes at the lesion site . ( a ) duplex containing the s - cdgdt base pair , viewed from the minor groove . ( b ) duplex containing the s - cdgdt base pair , viewed from the major groove . ( c ) duplex containing the s - cdgda base pair , viewed from the minor groove . ( d ) duplex containing the s - cdgda base pair , viewed from the major groove . base pairing and base stacking of the refined structures of the s - cdg containing duplex at the lesion site . the nonexchangeable protons were assigned based upon the sequential connectivity of the base proton h6 or h8 dipolar couplings with h1 deoxyribose protons ( figure 2c , d).for the modified strand , the noe connectivity was observed from g to c. the connectivity exhibited an interruption at x due to the lack of a proton at the c8 carbon . the x h1 proton was identified at 6.12 ppm ; it exhibited a weak x h1 t h6 noe , suggesting that the distance between these two protons was greater than in b - dna . the sequential connectivity resumed from t to t. for the modified strand , the deoxyibose h1 protons were observed within a narrow chemical shift window , between 5.8 and 6.3 ppm . the resonances of a h2 and a h2 appeared at 7.41 and 7.46 ppm , respectively . in addition , a h2 exhibited an noe with g n1h ( figure 4c ) , suggesting both a and a were intercalated . as expected , both h2 protons exhibited noes with h1 protons in the minor groove ( figure 9 ) . notably , a exhibited noes with both t h1 and a h1 of the 5-flanking ta base pair but did not exhibit noes with c h1 or g h1 of the 3-flanking cg base pair . expansion of the noesy spectrum ( 250 ms ) of the duplex containing the s - cdgda base pair showing the intercalation of a and a. noe cross - peaks are assigned as follows : a , t h6 t h1 ; b , t h6 x h1 ; c , a h2 a h1 ; d , a h2 t h1 ; e , a h2 a h1 ; f , a h2 x h1 ; g , a h2 a h1 ; and h , a h2 a h1. the assignments of x deoxyribose protons were made by analysis of scalar and dipolar couplings . figure 3b displays a tile plot derived from a noesy spectrum at 60 ms mixing time . x h1 exhibited dipolar couplings with h2 and h2 ; weak scalar couplings were also observed . h3 exhibited dipolar couplings with h2 , h2 , and h4 , whereas the scalar couplings were not observed . for the remainder of the duplex , the h2 , h2 , h3 , and h4 resonances were assigned unequivocally . the absolute configurations of the geminal h2 and h2 protons were assigned from their noes to h1 and h3. with the exception of the unresolved resonances for g , t , and c , h2 exhibited a weaker noe with h1 than did h2 , whereas it exhibited a stronger noe with h3 than did h2. the resonance assignments of the nonexchangeable dna protons are tabulated in table s5 in the supporting information . the resonances of the base imino protons were assigned based on sequential connectivity in noesy spectra and noes to the amino protons of watson crick base pairs ( figure 4c).the noe connectivity was observed from t g to g and from g t t t to g. the resonances of x n1h and t n3h were not assigned although a broad resonance was observed at 13.2 ppm at temperatures below 15 c . the assignment failed due to a lack of noe interactions . at the 5-neighbor base pair , g n1h exhibited noes with cn h1 and n h2 . with the exception of the terminal base pairs , the remaining noe cross - peaks arising from watson the scalar couplings of the 2-deoxyribose h1 protons with the h2 and h2 protons were measured from an e - cosy spectrum ( figure s1 in the supporting information ) . the jh1-h2 and jh1-h2 values for x were 2.7 and 7.2 hz , respectively . the jh4-h5 was 6.7 hz , whereas the jh3-h4 was not measurable . with the exception of the terminal nucleotides , the jh1-h2 for other nucleotides were 810 hz , and the jh1-h2 were 57 hz . the j coupling constants for the deoxyribose protons are tabulated in table s6 in the supporting information . h3 hmbc spectrum . with the exception of x , each exhibited a heteronuclear coupling with h3 of the 5-neighbor nucleotide the spectrum of the s - cdg - containing duplex was compared with the unmodified mismatched duplex ( figure s2 in the supporting information ) . at the modified nucleotide , the other p resonances were clustered within a modest chemical shift range , in the spectral region characteristic of b - dna . a total of 399 distance restraints , including 260 intranucleotide and 139 internucleotide restraints were calculated from the intensities of noe cross - peaks ( table s7 in the supporting information ) . a total of 30 noes involving the s - cdg protons were used as restraints . a total of 43 empirical distance restraints arising from watson crick base pairing interactions were used , as were 165 empirical torsion angle restraints that were applied to refine the nonterminal nucleotides . these were justified based upon nmr data , which suggested that structural perturbations were localized at and adjacent to the lesion site . the data suggested that no base pairing existed at the xa and ta base pairs , so base pairing restraints were not used for these base pairs . no torsion angle restraints were used for the cg , xa , and ta base pairs . ten rmd calculations , five each for a- and b - dna starting structures , were performed . the 10 emergent structures were minimized with respect to potential energy . all converged as indicated by pairwise rmsd comparisons ( table 1 ) . the accuracies of the emergent structures were evaluated by comparison of theoretical noe intensities calculated for the refined structure by the program corma to the experimental noe intensities to yield sixth root residuals ( r1 ) . these , as well as the residuals for intra- or internucleotide noes , were consistently less than 0.1 ( table 1 ) . r1 values for each nucleotide were less than 0.15 ( figure s3 in the supporting information ) . thus , the refined structures provided accurate depictions of the noe data . both x and a intercalated into the duplex ( figure 6 ) . consequently , the helical rise values from ct to xa and from xa to ta were greater than normal , 5.4 and 4.6 , respectively . figure 7c , d shows the base stacking and base pairing at the lesion site . significant perturbations in shift were observed from base pairs ta to ga , centered at the xa base pair . the ct base pair exhibited a greater than normal base pair twist of 54. the remaining base pairs exhibited normal base stacking ( table s8 in the supporting information ) . the s - cdg nucleotide was in the o4-exo , west pseudorotation ( figure 8b ) , with p = 264 and m = 47. the heavy atoms n9 , o3 , and c5 were axial about the deoxyribose ring . consequently , x h2 was farther from the x purine ring as compared to the h2 protons in b - dna , while c4 h2 was proximate to the x purine ring . with the exception of the terminal nucleotides , the six - membered ring c8n9c1o4c4c5 adopted the envelope ( half boat ) conformation . helicoidal analysis of the backbone torsion angles ( figure s4 in the supporting information ) showed that at the lesion site , the angle shifted from the characteristic 180 to 83. the angle shifted from 50 to 59. perturbations of the and torsion angles from 120 to + 157 and from 90 to 75 , respectively , were also observed . there was also a change for the n - glycosidic torsion angle from 120 to 143. . if not repaired , s - cdg blocks dna replication in e. coli and is genotoxic . in sos - induced e. coli , a mutation frequency of 34% is observed . most mutations are s - cdg a transitions , although s - cdg t transversions and a deletion of the 5-neighbor c are also observed . accordingly , structures in which s - cdg is placed opposite dt or da , representing intermediates leading to s - cdg a transitions and s - cdg t transversions , are of interest . the dg : dt mismatch often exists as a wobble base pair with both bases in the anti conformation . one might predict that locking s - cdg into the anti conformation about the n - glycosidic bond would be consistent with the formation of a wobble s - cdg : dt mismatch pair , and this appears to be the case . the formation of a wobble pair is consistent with the upfield shift of the x n1h resonances , which was also observed for the g n1h of the corresponding unmodified duplex ( figure 1b ) . however , the t n3h resonance was not observed , indicating enhanced solvent exchange at the s - cdgdt wobble pair . the structural refinement suggests the potential formation of a three - point hydrogen bond among x n1h , xnh2 , and to and a weak hydrogen bond between xo and t n3h ( figure 7 ) . however , the observation that the s - cdgdt wobble pair exhibits a tm 5 c lower than the corresponding duplex containing a dgdt mismatch pair ( figure 1a ) suggests that the incorporation of s - cdg reduces the stability of dgdt wobble pairing . the broadening of the t n3h and t n3h resonances as compared to the other thymine imino resonances ( figure 1b ) suggests that the greatest destabilization occurs at the modified xt and 3-neighboring ta base pairs . the base pair shifts at the cg and xt base pairs are consistent with this conclusion . similarly , for the s - cdg : dc pairing interaction , a 9 c decrease in tm was observed relative to the unmodified duplex . the thermal destabilization of this duplex is likely associated with the shift of the s - cdg deoxyribose to the o4-exo ( west ) pseudorotation , as opposed to the south pseudorotation ( c2-endo ) observed in b - dna or the north pseudorotation ( c3-endo ) in a - dna . moreover , the complementary t deoxyribose shifts to the c4-exo ( north ) pseudorotation , as evidenced by the jh1-h2 and jh1-h2 of 5.3 and 8.8 hz , respectively ( table s2 in the supporting information ) . the accommodation of the constrained s - cdg nucleotide necessitates helicoidal perturbation of the phosphodiester backbone torsion angles , , , and in the modified strand . additionally , smaller perturbations of the t phosphodiester backbone torsion angle in the complementary strand ( figure s4 in the supporting information ) may factor in the reduced stability of the s - cdgdt vs dgdt mispairing interaction . both s - cdg and da are inserted into the duplex , but they do not engage in hydrogen bonding . instead , helicoidal perturbations of the modified strand allow both to intercalate , creating a gap at the mismatched region . the absence of the g n1h a h2 noe agrees with the gap between a and g caused by the intercalation of s - cdg ( figure 6c , d ) . this is also consistent with the observation that a h2 exhibits noes with both h1 protons of the 3-flanking ta pair , but not with the h1 protons of the 5-flanking cg pair , suggesting a was close to ta but further from cg ( figure 9 ) . the observation of the a h2 a h2 noe suggests a remains intercalated . the 8 c decrease of the tm as compared to the duplex containing a dgda mispair is probably related to alterations of the s - cdg phosphodiester backbone torsion angles , , , and and to perturbations of the base pair shift parameters at the cg and xa base pairs , which are necessitated to accommodate the constrained s - cdg o4-exo ( west ) pseudorotation . non - natural , non - hydrogen - bonding base pair that demonstrates excellent polymerase activity , with slower rates of extension . the s - cdgda mismatch is distinct from the dgda mismatch as dgda mismatch pairs are influenced by sequence and ph . the failure to observe the imino resonances for base pairs cg , xa , and ta of the unmodified duplex is consistent with this notion and suggests an increased rate of exchange of these protons with solvent , perhaps accompanied by structural disorder . the dg(anti)da(syn ) pair has been identified in the crystalline state at ph > 7 , while the protonated dg(syn)da(anti ) pair has been identified at ph 6.6 . in solution , the dg(anti)a(anti ) pair is observed at neutral or basic ph conditions . another type of dg(anti)da(anti ) pairing is associated with tandem dg : da mismatches . these differences between the s - cdgda and dgda mismatches are attributed to the fact that the s - cdg lesion is locked into the anti conformation about the n - glycosidic bond and the shift of the deoxyribose to the o4-exo ( west ) pseudorotation . the da(anti)dg(anti ) face - to - face conformation would predict an noe between g n1h and a h2 , which is not observed ( figure 4c ) . the wobble s - cdgdt pair ( figure 6 ) is consistent with the site - specific mutagenesis studies in sos - induced e. coli , showing a preponderance of s - cdg da transition mutations . because low levels of s - cd g dt transversions are observed in e. coli , we surmise that low levels of datp are incorporated opposite s - cdg during trans - lesion synthesis . it has been suggested that klenow dna polymerases insert datp opposite s - cdg . further studies of templateprimers containing the s - cdg lesion complexed with error - prone polymerases will be of interest . the low levels of s - cdg dt transversions might reflect the distortion of the s - cdgda mismatch , in which both s - cdg and da are intercalated but do not hydrogen bond ( figure 6 ) . have reported that a non - natural , non - hydrogen - bonding base pair exhibiting a similar structure in duplex dna demonstrates excellent polymerase activity , with slower rates of extension . the structures of s - cdg placed opposite da or dt were determined and compared with the structure when s - cdg placed opposite dc . the s - cdgdt base pair adopted a hydrogen - bonded wobble conformation , while the s - cdgda base pair differed from dgda mispairs both s - cdg and da were intercalated , but no hydrogen bonding was observed . in each instance , the s - cdg deoxyribose adopted the o4-exo ( west ) pseudorotation and was accommodated by backbone and base - pairing helicoidal perturbations . collectively , these perturbations may be important in understanding the mutagenicity and genotoxicity of s - cdg .

although carbuncles are commonly seen and may heal on their own or respond well to treatment , in rare conditions , bacteria from carbuncles can spread into the bloodstream and migrate to other areas of the body . herein , we report on an elderly female who suffered from forehead carbuncle with intractable headache , later confirmed as having subgaleal abscess . physicians should pay special attention to elderly and immune - compromised patients with carbuncles located on the middle of the face , especially when accompanied by intractable headache , to avoid poor outcome . diastereomeric 8,5-cyclopurine 2-deoxynucleosides , containing a covalent bond between the deoxyribose and the purine base , are induced in dna by ionizing radiation . they are suspected to play a role in the etiology of neurodegeneration in xeroderma pigmentosum patients . if not repaired , the s-8,5-cyclo-2-deoxyguanosine lesion ( s - cdg ) induces pol v - dependent mutations at a frequency of 34% in escherichia coli . most are s - cdg a transitions , suggesting mis - incorporation of dttp opposite the lesion during replication bypass , although low levels of s - cdg t transversions , arising from mis - incorporation of datp , are also observed . we report the structures of 5-d(gtgcxtgtttgt)-35-d(acaaacaygcac)-3 , where x denotes s - cdg and y denotes either da or dt , corresponding to the situation following mis - insertion of either dttp or datp opposite the s - cdg lesion . the s - cdgdt mismatch pair adopts a wobble base pairing . this provides a plausible rationale for the s - cdg a transitions . the s - cdgda mismatch pair differs in conformation from the dgda mismatch pair . for the s - cdgda mismatch pair , both s - cdg and da intercalate , but no hydrogen bonding is observed between s - cdg and da . this is consistent with the lower levels of s - cdg t transitions in e. coli .

the collective arrangement of the teeth in function is quite important and has been subjected to a great deal of analysis and discussion over the years . as the mandible moves laterally , the lower posterior teeth leave their centric contact with the upper teeth and travel sideways down a path dictated by the condyles in the back and by the lateral anterior guidance in the front . in the diversified literature on occlusion and its role for functional patterns of the masticatory system , two concepts stand out : ( 1 ) canine protection as described by damico is said to favor a vertical chewing pattern and to prevent wear of teeth , as in lateral occlusion where the canine guides the mandibular movement directly through contact or indirectly through periodontal receptors . ( 2 ) group function as described by beyron following his observations on australian aborigines implies contact and stress on several teeth in lateral occlusion and indicates abrasion as a positive and inevitable adjustment . the reason for bringing any teeth into lateral function is to distribute stress and wear over more teeth . group function occlusion , which is also commonly known as unilateral balanced occlusion , is a widely accepted and used method of tooth arrangement in restorative dental procedures today . , the contacting inclines must be perfectly harmonized to border movements of the condyles and the anterior guidance . kleinberg has pointed out the importance of the feedback mechanism between mechanoreceptors in the temporomandibular joint and the functional mandibular movement pattern . in addition , a probable change in the input signals from the periodontal receptors after occlusal adjustment seems to affect the functional movement of the mandible . ingervall recorded tooth contact patterns in laterotrusion , protrusion of the mandible , and in the retruded position in young men with varying types of occlusion . contact on the nonfunctional side was found in half of the subjects in a 1.5-mm laterotrusive position and in one third of them in a 3-mm laterotrusive position . yaffe & ehrlich recorded the dynamic contact pattern of teeth in lateral glide movement in 72 individuals , 19 to 35 years of age , with normal tooth alignment and angle s class i molar and canine relationship . the lateral glide movement was divided into three stages to simulate the total range of events in lateral glide movement naturally demonstrated by the patient . this study has shown that lateral glide movement is a complex movement in which the nature of tooth contact is altering in location , direction , and number of teeth participating . consequently the restoration of an occlusion in accordance with a given concept does not always apply to all patients . the present study was designed to evaluate the incidence of occlusal patterns in a group of dental students . fifty subjects ( male : 27 , female : 23 ) aged 2029 years ( mean , 24.1 years ) were included in the study . they were selected from a group of 225 undergraduate students of the faculty of dentistry , tabriz university of medical sciences . the research and ethics committee approved the study protocol and informed consent was obtained from the subjects after they were provided an explanation of the general nature of the study . the criteria for selection were as follow : all subjects ( i ) were in their twenties ; ( ii ) had normal occlusal alignment , no temporomandibluar signs and symptoms with angle s class i relationship ; ( iii ) had full dentition except for third molars ; ( iv ) had no history of orthodontic therapy ; and ( v ) had no restorations involving a cusp . the occlusal contacts were recorded with occlusion foil ( 8 m thick , occlusions pruf - folie , ghm , germany ) in three lateral excursions , after laterally sliding the mandibular incisal point on both sides , 1 , 2 and 3 mm from the maximum intercuspation ( mi ) . when recording the occlusal contacts , each patient was instructed to close in the intercuspal position and to slide the mandible laterally to the right and left side performing the three lateral excursive movements designed as lateral 1 ( 1 mm from mi ) , lateral 2 ( 2 mm from mi ) , and lateral 3 ( 3 mm from mi ) on each side . each subject was required to sit upright in a dental chair with the frankfort plane almost horizontal . marks were made on the upper central incisors with a sharp black water - resistant pencil from the mandibular midline . three lines were marked on the maxillary incisor . in case of deviation of mandibular midline , a reference line was traced at the labial surface of the mandibular incisor in the intercuspal position to serve as a guide for the measurement . the blue occlusion foil was placed on the occlusal surface of the left - side most posterior mandibular molar , and the subject was requested to close his / her mandible to the mi . while a constant pulling force was maintained on the occlusion foil , red occlusion foil was put and the subject was requested to perform gliding movement to the right with the teeth in light contact . when the subject s mandible moved 1 mm right from the intercuspal position , the presence or absence of an occlusal contact was examined . only the red marks on teeth were considered to indicate occlusal contact . to prevent movement with mandibular opening or movement without occlusal contact and lateral - protrusive excursion , the movement was observed closely , and occasionally the subject was instructed to correct the movement . when the subject could not perform the movement voluntarily , he or she was asked to practice with the use of a hand mirror . the examination was continued from the left - side most posterior molar to the left - side central incisor sequentially . for examination of the molars , the occlusion foil was placed on both the mesial and distal sides of the occlusal surface . the same procedure was performed for the 1 , 2 and 3 mm right positions and also for the 1 , 2 and 3 mm left positions . in addition , the working - side occlusal contacts were recorded in the same manner . all recordings were performed by the same examiner and were repeated . in the case of differing results , all recordings were made between 10 oclock in the morning and 4 oclock in the afternoon to avoid possible diurnal variations . working - side occlusal contact patterns were determined for the total range of lateral positions and classified into three groups : canine protection , group function or others ( occlusal patterns other than those described ) . canine protection was defined as the contact of only working - side maxillary and mandibular canines in the total range of lateral positions from 1 to 3 mm . group function was defined as the contacts of two or more working - side teeth in at least one lateral position , and/or as single tooth contacts on the working - side in different lateral positions , e.g. the contact of only first molars in the 1 mm position followed by the contact of only canines in the 2 and 3 mm positions . the others type was identified when a contact pattern other than those described above was observed , e.g. contact of only first premolars throughout the lateral positions or contact patterns with no working - side contacts . figure 1 shows the number of working occlusal contacts recorded for each dental unit on right and left sides . the working - side occlusal contacts were mostly on the first premolar in r1 ( right 1 mm ) followed by canine ; but in l1 ( left 1 mm ) , the percentage of contacts on canine and first premolar were the same . the frequency of contact on premolars decreased gradually from the 1 to 3 on both sides . in all lateral positions , the frequency of contact decreased from the canine to the first molar as the tooth type became located posteriorly . however , the contact on the first molar almost was as prevalent as that on the second premolar . the frequency of the working - side canine contact increased from the 1 to the 3 mm position . figure 2 presents the number of non - working occlusal contacts recorded for each dental unit on right and left sides . the non - working - side contacts primarily involved the first and second molars , most frequently the second molar . the prevalence of non - working - side contacts decreased as the mandible moved from the 1 to 3 mm position on the premolars and molars . table 1 shows the percentage of occlusal patterns in mandibular lateral movement on the right and left sides . most of working - side contact patterns were classified as group function ( 60% ) ; canine protection was rare ( 17.3% ) , and contact patterns other than canine protection and group function were found in 23.7% of the contact patterns on the right side . on the left side , group function was found in 51.3% , canine protection in 20.7% and others in 28% of the contact patterns . most of the subjects ( 66% ) had the same pattern on both sides : 42% group function , 8% canine protection , and 16% others . this study demonstrated that the occlusal contact pattern during lateral movement differs between 1 mm , 2 mm and 3 mm . in the position close to the maximum intercuspation the occlusal contact pattern varying with lateral positions should be a critical factor on diagnosis and treatment of occlusal disharmonies . the marked difference in the pattern of occlusal contacts between the 1 , 2 and 3 mm positions , suggests that the contact pattern in the 1 mm position reflects a pattern of occlusal contact distinct from that commonly observed during lateral excursion . in a study analyzing two working - side inter - occlusal contacts , a large number of subjects presented unclassified patterns of articulations . more individuals had canine guidance on the left side , whereas the most frequent pattern on the right side was group function . another study on occlusal contact areas using a 3-d digitization measurement system found that , at intercuspal position , estimated occlusal contact areas were 12.6 mm . however , after 3.0 mm of lateral excursion , their areas were sharply reduced to 2.2 mm . ogawa et al10 - 12 found most contact patterns belonged to group function and a few to canine protection . they also found the presence of occlusal contacts in different lateral positions may have different effects on biomechanics of the related teeth . for instance , the force on individual teeth may be more traumatic in the more lateral positions because of the increased vector of the force . occlusal force is transmitted to the teeth during four functional and parafunctional stages : mastication , swallowing , clenching and grinding . the occlusal contact during mastication and swallowing is suggested to occur mainly in lateral positions close to the mi , i.e. within 1 mm of the mi.10 - 1 in addition , forces during these two functional movements are relatively low and their durations are short . taking into consideration these various factors , the biomechanical significance of non - working - side occlusal contact may increase over the non - functional range of lateral movement corresponding to the 3 mm position . in the present study , incidence of non - working - side occlusal contact decreased from 1 to 3 mm ; so it does not seem appropriate to deduce that contact in this position produces harmful force to the teeth or periodontal structures . however , this does not exclude the possibility that the non - working - side contacts in the position close to the mi may be important in masticatory function . contact patterns other than canine protection and group function were found in 23% of the contact patterns on the right side . on the left side , group function was seen in 51% , canine protection in 21% and others patterns in 28% of the studied subjects . on laterotrusion , most subjects had group function on the working side but canine protection was rare . zinc deficiency is an important cause of morbidity due to infectious diseases and growth - faltering among young children . increased demand of zinc due to rapid growth and decreased intake of zinc due to inadequate feeding practices predispose preschool children , especially living in communities of low socioeconomic level at an elevated risk of zinc deficiency ( 1 ) . compelling evidence from intervention trials suggests that supplementation of zinc could reduce the risk of pneumonia and the risk and duration of diarrhoea , dysentery , and malaria among preschool children ( 25 ) . however , little information is available on the global prevalence of zinc deficiency . on the other hand , the population / national - level zinc - supplementation programmes have achieved limited success in alleviating zinc deficiency worldwide ( 6 ) . lack of reliable methods to determine the levels of plasma zinc , paucity of knowledge of the socioeconomic , dietary and demographic factors influencing the distribution of zinc deficiency , and inadequate documentation of rates and causes of zinc deficiency among vulnerable preschool children and its impact on childhood morbidities among different populations are some most important limiting factors ( 78 ) . traditionally , zinc deficiency at the population level has been estimated by the surrogate markers , such as total daily per - capita amount of zinc in the food - supply , compared to the likely zinc requirements , or by the rates of stunting among preschool children ( 9 ) . using the food balance - sheets , wuehler et al . estimated that about 71.2% of the total population in southeast asia was at risk of developing zinc deficiency ( 10 ) . the prevalence of zinc deficiency among a population can be assessed by a combination of techniques , such as the prevalence of clinical outcomes of zinc deficiency ( diarrhoea , pneumonia , stunting ) , assessment of intake of dietary zinc , biochemical measures of zinc concentration , or assessment of functional outcomes of zinc supplementation ( 11 ) . a community - based study conducted in the north - west frontier province in pakistan reported that one in two preschool children suffers from zinc deficiency ( 12 ) . zinc deficiency has been suspected to be prevalent among preschoolers in india due to high consumption of cereal - based weaning diets and high rates of recurrent infections . there is also limited systematically - collected information on the quantitative estimates of zinc deficiency , especially those living in communities of low socioeconomic level ( 13 ) . determining the magnitude of zinc deficiency and its association with risk of morbidities among preschool children in india can contribute to planning strategies to alleviate zinc deficiency and the related adverse health consequences . in this study , we present the descriptive epidemiology of zinc deficiency and the associated risk of morbidity due to infectious diseases among preschool children in an urban slum in northern india . the main study was a double - masked , randomized controlled zinc - supplementation trial carried out in an urban slum in new delhi , and the details of the study population , design , methods , eligibility , recruitment , and randomization have earlier been reported ( 1415 ) . briefly , 940 children aged 635 months and presenting to the community - based clinic with history of acute diarrhoea ( defined as having passed at least four unformed stools in the past 24 hours and having diarrhoea for less than seven days ) were enrolled and randomly allocated to either zinc group ( n=468 ) or control group ( n=472 ) . a randomization schedule with permuted blocks of fixed length ( 10 per block ) was employed for randomization . at the first home - visit , after recovery from diarrhoeal episode at enrollment ( passage of three or fewer stools for three consecutive days ) , 609 of these children were followed up for six months ( long follow - up ) , and for the rest , the follow - up was terminated after recovery from diarrhoeal episode ( n=331 ) . home - visits daily for supplementation and every fifth day for the assessment of morbidity were made to evaluate preventive effects of zinc on diarrhoea , dysentery , and respiratory disease - related morbidity . the prevalence and descriptive epidemiology of zinc deficiency were based on the baseline evaluation of the enrolled children ( n=940 ) . of the 609 children in the long follow - up , 116 allocated to the control group belonging to the upper and the lower 25 quartile of baseline plasma zinc status were selected for evaluating the association of zinc deficiency with prospective morbidity . the prospective morbidity was assessed for a follow - up period of 120 days . for comparison of the prevalence of zinc deficiency among children with acute diarrhoea and those without diarrhoea , 193 children from the control group who did not have diarrhoea in the three days before the collection of blood samples after 120 days of supplementation ( fig . schematic representation of study design at enrollment , a health worker collected socioeconomic and demographic data , information on the breastfeeding status , intake of non - breastmilk , frequency of consumption of zinc - rich foods ( such as green - leafy vegetables , pulses , rice , wheat bread , fruits , meat , fish , eggs , chicken , butter , etc . ) in the two weeks before enrollment and recorded anthropometric measurements . a research physician gathered a detailed morbidity history ( information regarding the episodes and duration of diarrhoea , vomiting in the last 24 hours , pneumonia , and acute respiratory tract infections [ alris ] in the last two months ) , and nutritional profile and conducted a thorough physical examination . blood sampling was done to assess the levels of plasma zinc at the baseline ( n=940 ) and after 120 days of follow - up ( n=472 ) . for the estimation of plasma zinc , venous blood sample was collected in a monovette trace element - free heparinized syringe ; the plasma was separated within 15 minutes of collection of the samples and was transferred into trace element - free eppendorf plastic tubes to be stored at 20 c . c - reactive protein was also estimated on a cobas fara analyzer ( roche products , welwyn , united kingdom ) . children were prospectively followed up by home - visits every fifth day by the health worker . using the standard definitions of the world health organization , information was gathered on days of illness , days and episodes of diarrhoea , days of dysentery , days with alri , and episodes of pneumonia . in the case of any morbidity , the children were referred to the project physician for management . for evaluating the prevalence of zinc deficiency among the study population , two cut - offs were used : plasma zinc < 60 g / dl and plasma zinc < 70 g / dl . to understand the descriptive epidemiology of zinc deficiency , we categorized the entire cohort of 940 children by baseline plasma zinc values into lower quartile ( baseline plasma zinc values below 25 percentile , i.e. plasma zinc 56.0 g / dl ) , middle quartile ( baseline plasma zinc values 25 percentile , and 75 percentile , i.e. plasma zinc > 56.0 to 70.0 g / dl ) , and upper quartile ( baseline plasma zinc values > 75 percentile , i.e. plasma zinc > 70.0 g / dl ) and compared the baseline socioeconomic , demographic and dietary factors affecting the levels of plasma zinc among the quartiles . to evaluate the impact of baseline plasma zinc on prospective morbidities , the risk of morbidities was compared among the lower and upper quartile groups after adjusting for covariates , such as socioeconomic score , education of mothers / fathers , water supply , age and gender of the child . person - time analysis was performed with actual follow - up as denominator . for the effect on the incidence of diarrhoea / dysentery , respiratory and other childhood illnesses all statistical analyses were carried out using the spss software ( version 12.0 ) ( spss inc . , chicago , illinois , usa ) and the stata software ( version 9.2 ) ( stata corp . the main study was a double - masked , randomized controlled zinc - supplementation trial carried out in an urban slum in new delhi , and the details of the study population , design , methods , eligibility , recruitment , and randomization have earlier been reported ( 1415 ) . briefly , 940 children aged 635 months and presenting to the community - based clinic with history of acute diarrhoea ( defined as having passed at least four unformed stools in the past 24 hours and having diarrhoea for less than seven days ) were enrolled and randomly allocated to either zinc group ( n=468 ) or control group ( n=472 ) . a randomization schedule with permuted blocks of fixed length ( 10 per block ) was employed for randomization . at the first home - visit , after recovery from diarrhoeal episode at enrollment ( passage of three or fewer stools for three consecutive days ) , 609 of these children were followed up for six months ( long follow - up ) , and for the rest , the follow - up was terminated after recovery from diarrhoeal episode ( n=331 ) . home - visits daily for supplementation and every fifth day for the assessment of morbidity were made to evaluate preventive effects of zinc on diarrhoea , dysentery , and respiratory disease - related morbidity . the prevalence and descriptive epidemiology of zinc deficiency were based on the baseline evaluation of the enrolled children ( n=940 ) . of the 609 children in the long follow - up , 116 allocated to the control group belonging to the upper and the lower 25 quartile of baseline plasma zinc status were selected for evaluating the association of zinc deficiency with prospective morbidity . the prospective morbidity was assessed for a follow - up period of 120 days . for comparison of the prevalence of zinc deficiency among children with acute diarrhoea and those without diarrhoea , 193 children from the control group who did not have diarrhoea in the three days before the collection of blood samples after 120 days of supplementation ( fig . at enrollment , a health worker collected socioeconomic and demographic data , information on the breastfeeding status , intake of non - breastmilk , frequency of consumption of zinc - rich foods ( such as green - leafy vegetables , pulses , rice , wheat bread , fruits , meat , fish , eggs , chicken , butter , etc . ) in the two weeks before enrollment and recorded anthropometric measurements . a research physician gathered a detailed morbidity history ( information regarding the episodes and duration of diarrhoea , vomiting in the last 24 hours , pneumonia , and acute respiratory tract infections [ alris ] in the last two months ) , and nutritional profile and conducted a thorough physical examination . blood sampling was done to assess the levels of plasma zinc at the baseline ( n=940 ) and after 120 days of follow - up ( n=472 ) . for the estimation of plasma zinc , venous blood sample was collected in a monovette trace element - free heparinized syringe ; the plasma was separated within 15 minutes of collection of the samples and was transferred into trace element - free eppendorf plastic tubes to be stored at 20 c . c - reactive protein was also estimated on a cobas fara analyzer ( roche products , welwyn , united kingdom ) . children were prospectively followed up by home - visits every fifth day by the health worker . using the standard definitions of the world health organization , information was gathered on days of illness , days and episodes of diarrhoea , days of dysentery , days with alri , and episodes of pneumonia . in the case of any morbidity for evaluating the prevalence of zinc deficiency among the study population , two cut - offs were used : plasma zinc < 60 g / dl and plasma zinc < 70 g / dl . to understand the descriptive epidemiology of zinc deficiency , we categorized the entire cohort of 940 children by baseline plasma zinc values into lower quartile ( baseline plasma zinc values below 25 percentile , i.e. plasma zinc 56.0 g / dl ) , middle quartile ( baseline plasma zinc values 25 percentile , and 75 percentile , i.e. plasma zinc > 56.0 to 70.0 g / dl ) , and upper quartile ( baseline plasma zinc values > 75 percentile , i.e. plasma zinc > 70.0 g / dl ) and compared the baseline socioeconomic , demographic and dietary factors affecting the levels of plasma zinc among the quartiles . to evaluate the impact of baseline plasma zinc on prospective morbidities , the risk of morbidities was compared among the lower and upper quartile groups after adjusting for covariates , such as socioeconomic score , education of mothers / fathers , water supply , age and gender of the child . person - time analysis was performed with actual follow - up as denominator . for the effect on the incidence of diarrhoea / dysentery , respiratory and other childhood illnesses all statistical analyses were carried out using the spss software ( version 12.0 ) ( spss inc . , chicago , illinois , usa ) and the stata software ( version 9.2 ) ( stata corp . zinc deficiency as assessed by estimation of plasma zinc showed that 73.3% of the children were zinc - deficient ( plasma zinc < 70 g / dl ) , of whom 33.8% had levels of plasma zinc below 60 g / dl . younger children were at a lower risk of zinc deficiency than were older children ; however , the prevalence was similar among male and female children ( table 1 ) . prevalence of zinc deficiency among preschool children the descriptive epidemiology of zinc deficiency is presented in table 2 . the children in the cohort were categorized into three quartiles based on levels of baseline plasma zinc . the nutritional status , breastfeeding status , recent intake of dietary zinc - rich food(s ) , including the consumption of non - breastmilk , and socioeconomic level among children were similar across the quartiles . the prevalence of zinc deficiency among children having acute diarrhoea at baseline was similar to those without diarrhoea at the end of the study ( fig . 2 ) . association of baseline zinc levels with child characteristics * lower 25 percentile plasma zinc 56.0 g / dl ; middle 50 percentile ( 25 percentile and 75 percentile ) plasma zinc > 56.0 g / dl and plasma zinc 70.0 g / dl ; n=110 , 199 , and 81 in age 12 year(s ) ; n=47 , 91 , and 29 in age > 2 years ; figures in parentheses indicate percentages ; alri = acute lower respiratory infection ; ses = socioeconomic status comparison of prevalence of zinc deficiency among children with acute diarrhoea and those without diarrhoea after 120 days of follow - up , children in the lower quartile ( based on levels of plasma zinc ) suffered a significant relative increase in the risk of days of illness by 15% ( 95% confidence interval [ ci ] 822 ) , days of diarrhoea by 22% ( 95% ci 937 ) , days with dysentery by 44% ( 95% ci 12135 ) , days with alri by 49% ( 95% ci 1692 ) , and pneumonia episodes by 5% ( 95% ci 4391 ) compared to children in the upper quartile . risk ratios did not change after adjusting for literacy of mothers , literacy of fathers , score of socioeconomic status , source of water supply , age , and gender ( table 3 ) . association of baseline zinc levels with prospective morbidity in 120-day follow - up comparison of lower 25 quartile vs upper 25 quartile * lower 25th percentile plasma zinc 56.0 g / dl ; * * upper 25th percentile plasma zinc > 70.0 g / dl ; adjusted for literacy of father , literacy of mother , socioeconomic status , water supply , age , and gender ; alri = acute lower respiratory tract infection ; ci = confidence interval ; rr = relative risk our study documents the epidemiology and magnitude of zinc deficiency among preschool children at the community level in an urban slum in india . it also describes the association between the levels of plasma zinc and the risk of morbidities due to infectious diseases . recent studies , using data on food - availability , estimated that zinc deficiency affects about one - third of the world 's population , with estimates ranging from 4% to 73% across subregions . although severe zinc deficiency is rare , mild - to - moderate zinc deficiency is quite common throughout the world ( 17 ) . the prevalence of zinc deficiency found in our study concurs with the results of the community - based study carried out in pakistan ( 12 ) and reflects the distressing magnitude and distribution of zinc deficiency . the high prevalence of zinc deficiency observed in the present study could be explained by the low bioavailability of zinc from cereal - based diets and increased rate of subclinical infection ( 18 ) . there is a lack of agreement on the normal variation in plasma zinc values according to age . found no age - dependent variation in total serum zinc among healthy dutch infants and children ( 19 ) . likewise , karr et al . used atomic absorption spectrophotometer to estimate the plasma zinc values in healthy preschool australian children and found no significant age - dependent variation ( 20 ) . however , the results of our study showed a higher proportion of children in the upper quartile ( based on levels of plasma zinc ) among the 611 months age - group compared to children in the higher age - groups . this could be due to appropriate breastfeeding practices among younger age - group which might lead to better zinc status . recent studies have shown that zinc status is influenced by consumption of test - meal among healthy adult volunteers ( 19 ) . on the other hand , bitarak wate et al . found no significant association between the nutritional status and the levels of serum zinc ( 21 ) . likewise , in our study , we observed that neither the breastfeeding status nor the consumption of zinc - rich foods , or consumption of non - breastmilk during the two weeks preceding enrollment had any significant impact on the levels of plasma zinc . although the effects of poor intake and increased micronutrient demands are well - described , the potential effects of acute infections on the body 's micronutrient status are less well - understood . there is little information on the short - term compartment changes of several micronutrients , including zinc following an infection ( 22 ) . a recent study by strand et al . reported a decline in concentrations of plasma zinc during episodes of acute diarrhoea ( 23 ) . previously , chaudhary et al . reported a significant fall in plasma zinc after acute and persistent diarrhoea in indian children ( 24 ) . similarly , bitarakwate et al . found that concentration of zinc in serum of children with persistent diarrhoea was significantly lower than that of children without diarrhoea ( 21 ) . however , our results showed that the prevalence of zinc deficiency ( plasma zinc < 70.0 g / dl ) at baseline when all the children had acute diarrhoea was comparable with the prevalence in the second sample among the children who did not have diarrhoea three days before the last blood - sampling . this could be due to the homeostatic control mechanism in the body that shifts zinc stores in the body during diarrhoea to balance the loss through excretion . there is evidence to show preventive and therapeutic effects of zinc supplementation on various childhood morbidities ( 18,2530 ) . our morbidi - ty - related results showed that children with zinc deficiency were at a higher risk of morbidities compared to zinc - sufficient children . these results substantiate the findings of a limited number of previously - published studies conducted in similar population settings relating the zinc levels to prospective morbidities ( 3132 ) . although our study children had acute diarrhoea at baseline , the findings are comparable as we have shown that the prevalence of zinc deficiency during diarrhoea was similar to diarrhoea - free days . the limitations of the study include that zinc deficiency was assessed using plasma zinc only and the intake of dietary zinc was not assessed . in conclusion , this study provides systematic information regarding the factors influencing the zinc levels among preschool children in low socioeconomic settings and substantiates the relationship between zinc levels and prospective morbidities . the authors acknowledge the contributions of parents of the study children , study team , including health workers , supervisors , physicians , technicians , data management , and other support staff . the authors also acknowledge contributions of the world health organization and the thrasher research fund for funding the study .

background and aims this study observed occlusal contacts and their area on the teeth during lateral mandibular movements . the percentage of each occlusal pattern was determined . materials and methods fifty subjects ( male : 27 , female : 23 ) , aged 20 - 29 years , were included in the study . the criteria for selection were as follow : all subjects ( i ) were in their twenties ; ( ii ) had normal occlusal alignment , no temporomandibular signs and symptoms with angle s class i relationship ; ( iii ) had full dentition except for third molars ; ( iv ) had no history of orthodontic therapy ; and ( v ) had no restorations involving a cusp . the occlusal contacts were recorded with occlusion foil in three lateral excursions : 1 , 2 and 3 mm from the maximum intercuspation . data were analyzed with chi - square test . results most of working - side contact patterns were classified as group function ( 60% ) . canine protection was rare ( 17% ) . contact patterns other than canine protection and group function were found in 23% of the contact patterns on the right side . on the left side , group function was seen in 51% , canine protection in 21% and others patterns in 28% of the studied subjects . conclusion on laterotrusion , most subjects had group function on the working side but canine protection was rare . community - based data relating to factors influencing zinc deficiency among preschool children in india are inadequate . data of a large , double - blinded , randomized , controlled zinc - supplementation trial were used for assessing the descriptive epidemiology of zinc deficiency among children aged 635 months ( n=940 ) . in total , 609 children were followed up for 120 days for information on morbidity . of these children , 116 from the control group belonging to the upper and the lower 25th quartile of plasma zinc status at baseline were selected for assessing the association of zinc deficiency with prospective morbidity . at baseline , demographic , socioeconomic and dietary information was collected , and anthropometric measurements and levels of plasma zinc were assessed . at baseline , 73.3% of the children were zinc - deficient ( plasma zinc < 70 g / dl ) , of which 33.8% had levels of plasma zinc below 60 g / dl . a significantly higher risk of morbidity was prevalent among the subjects with lower plasma zinc compared to those with higher levels of plasma zinc .

intradural disc herniation ( idh ) is a rare disease and its incidence is reported as 0.26 - 0.30% of all disc herniations16 ) . idhs primarily occur as a result of chronic degenerative diseases and rarely occur due to traumatic events1,14 ) . traumatic lumbar spine lesions usually result in bony fractures and paraspinal soft tissue injuries rather than isolated disc herniations . in this report , we present a rare case of a patient who exhibited an intradural mass lesion at l1 , which initially mimicked a spinal subdural hematoma ( sdh ) and was later determined to be an idh without an accompanying lumbar spine bony fracture after a traumatic event . a 52-year - old man presented at the hospital with numbness in his left calf and ankle after falling accident two days ago . the patient was initially admitted at another medical center after the accident but transferred to our hospital at his request . a motor power grade was intact with slightly decreased sensation along the left l5 dermatome . bladder and bowel functions were intact , and no other significant abnormalities were found upon neurological examination . in his medical history , he had visited our hospital six weeks ago with complaints of radiating pain in his right leg along the l5 dermatome after falling down from 2 m height . at that time , his lumbar spine magnetic resonance image ( mri ) scan exhibited diffuse bulging intervertebral disc in l4 - 5 ( fig . 1 ) . the patient had received a conservative treatment with a non - steroidal anti - inflammatory drug and discharged with the improvement of symptoms . at this time , the patient 's plain radiography did not present bony abnormalities such as fractures or dislocations . in his lumbar computed tomography ( ct ) scan , a small calcified lesion was found at the l1 - 2 level , posterior to the disc ( fig . 2 ) . on his lumbar t2-weighted mri , a high signal intensity with an amorphous shape lesion was located intradurally and it compressed the spinal cord anteriorly at the l1 level ( fig . 3c ) and it presented heterogeneous low signal intensity in the gradient echo ( gre ) image ( fig . we considered the lesion to be a spinal sdh , and decided to perform a conservative treatment . at the follow up mri scan after two weeks of treatment , the lesion still remained unchanged without any improvement of numbness , so we decided to operate . a subtotal laminectomy was performed at l1 , which revealed an out - punching whitish soft particle on the dorsal dura . the particle was thought to be a ruptured disc material and removed gently , then sent for pathology . the pathologic results determined the particle to be degenerating fibrocartilagenous tissue corresponding to an intervertebral disc . after removing the extradural lesion , a midline durotomy was done from the dura defect uncovering large , cartilaginous disc particles in the middle of the intradural space ( fig . , a hard bony lesion remained adhesively to the ventral dura and a small ventral dura defect was identified around the bony lesion ( fig . the hard bony lesion was thought to be a calcified lesion at the posterior l1/2 intervertebral disc in his lumbar spinal ct scan . we tried to repair the dura defect to protect a leakage of cerebrospinal fluid ( csf ) , but the ventral dura tear could not be repaired due to its severe adhesion . a watertight closure on dorsal dura after the operation , the patient 's numbness in his left leg has gradually improved . two weeks after surgery , the patient was discharged from the hospital with free of symptoms . idhs are rare clinical diagnoses that typically occur in patients aged in their fifties11 ) . dandy4 ) first reported an intradural lumbar disc herniation in 1942 and the incidence of idhs is reported between 0.04 and 0.33% of all reported lumbar disc herniations9 ) . the most common site of intradural lumbar disc herniations is the l4 - 5 ( 55% ) area , followed by l3 - 4 ( 16% ) , and l5-s1 ( 10% ) , far fewer occur at l1 - 2 and l2 - 311,13,15 ) . furthermore , traumatic idhs are rarer events and the incidence rate has not yet been reported in the literature . most traumatic disc herniations in the lumbar spine frequently have severe bony injuries such as fractures and dislocations8 ) . in our case , however , we found an idh after a traumatic event without fractures or dislocations . although the mechanisms that cause disc herniations to pass the dura mater are unclear , the widely accepted hypothesis states that adhesion between the ventral dura and posterior - longitudinal ligament ( pll ) leads to the successive perforation of these firmly adhesive tissues , including the annulus fibrosus , due to the increased intradiscal pressure1,4,9,10 ) . dandy4 ) explained that sudden pressure on the protruded disc might erode and then penetrate the overlying ventral dura . other reports have suggested that dense adhesion , whether it is congenital or acquired , fixate the dural sac1,14 ) . diagnosing an idh is difficult due to its rarity . diagnosis using radiological images is a difficult task , because lumbar idhs can have various radiological and clinical features3,17 ) . furthermore , lesions may be ignored in simple radiography and ct scans , which are one of the most frequently used radiologic examinations in the diagnosis of spinal injuries . hidalgo - ovejero et al.6 ) reported that the presence of epidural gas in a ct scan could be a clue of the presence of an idh . in our case , however , we could not find epidural gas on the ct scan . since wasserstrom et al.18 ) made the first idh diagnosis using a gadolinium - enhanced mri in 1993 , the rim enhancement of herniated discs has been accepted as a typical mri finding in idhs . furthermore , choi et al.2 ) presented the loss of pll continuity and a sharp beak - like feature on a t2-weighted image as indicators of idh . in our case , we also observed a peripheral rim enhancement pattern on the gadolinium - enhance t1 mri , but it was not enough to consider the idhs due to the small size of portion ( fig . moreover , the loss of pll continuity was not definite on our initial lumbar spine mri . therefore , our initial impression of the intradural mass at l1 was a spinal sdh , although a traumatic spinal sdh is also very rare12 ) . on mri , the spinal sdh yielded high signals on both t1 and t2 , but the signal intensity of the spinal sdh on the mri also depend on the duration7 ) . in our case , 3a , b ) and an isosignal on the t1 scan . on the gre scan , however , the intradural lesion had a high signal - intensity with a low signal void inside the intradural lesion ( fig . an intradural abscess could be considered as another differential diagnosis , but we excluded it because there were no signs of infection such as fever or leukocytosis . additionally , the patient had visited our hospital six weeks previously for radiating pain in his right lower leg . at that time , the patient checked a lumbar mri scan , and was diagnosed with a diffuse bulging disc in l4 - 5 and there was no intradural mass lesion . compared with the mri from that visit , de - novo intradural lesion was detected . moreover , the height and the signal intensity of the intervertebral disc of l1 - 2 had decreased in the recent study ( figs . we assumed that a strong vertical pressure had been applied to cause the intervertebral disc rupture , then the ruptured particles penetrated the pll , ventral dura , and through dorsal dura to the epidural space . according to the clues mentioned above , we could assume that the idh developed from a trauma rather than a degenerative condition . we experienced a rare case of a lumbar idh after trauma without an accompanying bony fracture or dislocation . for patients with an intradural lesion at the lumbar spine after trauma as with the patient in this report , idhs should be considered as differential diagnoses and prompt removal of the intradural mass is necessary . initially , they were considered as metastasis from an occult primary tumor in gonads , but now it is known that they arise either as a consequence of abnormal migration of germ cells during embryogenesis or from a different histogenetic origin . choriocarcinoma of liver without a detectable primary tumor in gonads , retroperitoneum or mediastinum is termed as primary hepatic choriocarcinoma . due to varied clinical presentations and extreme rarity of the tumor , diagnosis on small needle biopsy is difficult . we present a case of adult male diagnosed to have primary hepatic choriocarcinoma on histopathology and immunohistochemical studies . a 40-year - old male was referred to us with history of sudden onset pain in abdomen . he was admitted in private hospital where computed tomography ( ct ) scan of abdomen was done which was suggestive of ruptured haemangioma in left lobe of liver ( figure 1 ) . patient was resuscitated and given adequate blood transfusions and was referred to our centre . on admission abdominal exploration revealed about 1.5 to 2 l of haemoperitoneum and multiple nodular lesions in both lobes of liver with large ruptured lesions in left lobe of liver with active bleeding . after achieving adequate inflow and outflow control , left lateral segmentectomy was done in view of active bleeding ( figure 2 ) . histopathology showed atypical trophoblastic cells predominantly cytotrophoblast and few syncitiotrophoblast lying in sheets as well as in clusters with large number of mitotic figures along with the normal hepatocytes suggestive of high grade malignant tumor ( figure 3 ) . immunohistochemistry was positive for beta human chorionic gonadotrophin ( figure 4 ) and negative for ck , ema , cd-30 , afp , cd-31 and cd-34 suggestive of choriocarcinoma . his serum hcg levels were significantly raised however serum levels of afp , cea and ca 19 - 9 were normal . patient died on postoperative day 10 due to sudden cardiopulmonary arrest . on autopsy , multiple nodular lesions were present on the remaining liver . bilateral testes were grossly normal which on subsequent serial sectioning and histological examination did not show any evidence of pathologic features associated with germ cell tumor regression or a scar . based on histopathological report and autopsy findings figure 1computed tomography scan of abdomen showing ruptured haemangioma in left lobe of liver with haemoperitoneum . computed tomography scan of abdomen showing ruptured haemangioma in left lobe of liver with haemoperitoneum . figure 3atypical trophoblastic cells predominantly cytotrophoblast and few syncitiotrophoblast lying in sheets as well as in clusters along with the normal hepatocytes . atypical trophoblastic cells predominantly cytotrophoblast and few syncitiotrophoblast lying in sheets as well as in clusters along with the normal hepatocytes . majority of them are infantile type , may represent metastasis from an occult placental choriocarcinoma . primary hepatic choriocarcinoma in adults are known to arise from abnormal migration of germ cells during embryogenesis or different histogenetic origin . clinically , patient may present with right upper abdominal pain and/or abdominal lump or distension . those with advanced disease may present with symptoms due to metastasis to various organs like brain and lungs . infertility , gynaecomastia and features of thyrotoxicosis may present in some patients , attributed to over production of hcg by tumour cells . very rarely , it may rupture spontaneously producing haemoperitoneum and may present as an acute abdomen , as in our case . major issue in diagnosing primary hepatic choriocarcinoma is to exclude metastasis from an occult primary in gonads by serial sectioning and histological examination , as these tumors may be small or undergo spontaneous regression at the time of metastasis . diagnosis on small needle biopsy is difficult due to rarity of the tumor in liver . immunoshistochemical staining for beta hcg , plap , hpl , hepar-1 , may aid the diagnosis and helps to differentiate from the other tumours which mimic choriocarcinoma like giant cell variant of poorly differentiated hepatocellular carcinoma , or tumor with trophoblast like giant cells . finally imaging modalities like ultrasonography and ct scan may be useful to assess the extent of primary tumour and metastasis to various organs . treatment consists of complete surgical resection of tumor if localized to liver and without ascites followed by chemotherapy . for advanced or metastatic disease chemotherapy is given . are etoposide , methotrexate , actinomycin - d , cyclophosphamide . prognosis of primary hepatic choriocarcinoma is distinctly poor as compared to its testicular counterpart owing to lack of restrictive effect of tunica albugenia as in the testes ; hence they attain a large size and often invade adjacent vital structures by the time diagnosis is made . average survival being 2 to 8 months as reported in literature , hence further studies are needed for early diagnosis and better treatment to improve survival .

intradural lumbar disc herniation is a rare disease . according to the reports of intradural lumbar disc herniations , most cases have developed as a chronic degenerative disc diseases . traumatic intradural lumbar disc herniations are even rarer . a 52-year - old man visited our emergency center with numbness in his left calf and ankle after falling accident . initial impression by radiologic findings was a spinal subdural hematoma at the l1 level . a follow up image two weeks later , however , did not demonstrate any interval change . the patient was decided to have an operation . in operative findings , a ruptured disc particle penetrating the ventral and dorsal dura was indentified after laminectomy . it was assumed to be a traumatic outcome not a degenerative change . choricarcinoma is a beta human chorionic gonadotrophin secreting neoplasm pertinent to uterus and pregnancy mostly . it occurs primarily in gonads but rarely in extragonadal sites . primary hepatic choriocarcinoma is an extremely rare tumor . most of the reported cases are seen in infants representing metastasis from an occult placental choriocarcinoma . till date , only 7 cases of primary hepatic choriocarcinoma in adults have been reported in literature . we present a case of a 40-yearold male presenting as haemoperitoneum due to ruptured hepatic tumor . he underwent emergency left lateral segmentectomy . he died on 10th postoperative day . the surgical specimen and autopsy findings confirmed it to be primary hepatic choriocarcinoma . this is the first case report from indian subcontinent . a brief case report and review of literature is presented .

human ifn-2 ( il-28a ) is a relatively new cytokine , in which the genomic structure resembles that of the il-10 family , but the protein structure is more closely related to type i ifn than to interleukin- ( il- ) 10 [ 1 , 2 ] . for example , it can induce antiviral activity in cell lines , though the potency is weaker than other ifns , and has the potential antitumor effect against human lung cancer cells . it has also been discovered that ifn-2 is capable of exacerbating t - cell - mediated autoimmune diseases such as uveitis . treatment with ifn-2 completely halts and reverses the development of collagen - induced arthritis , dramatically reduces the numbers of proinflammatory il-17-producing th17 and t cells in the joints and inguinal lymph nodes , and restricts recruitment of il-1b - expressing neutrophils . however , ifn-2 seems not effective in inducing tr1 cells and can not induce proliferation of regulatory t cells from cord blood cd4(+ ) t cells . recently , it was found that the expression level of ifn-2 mrna was significantly increased during naturally occurring respiratory viral infections in children with asthma and that ifn-2 modulates lung dendritic cells ( dc ) function to promote th1 immune skewing and suppresses allergic airway disease . these suggest that ifn-2 is not only involved in autoimmune diseases but also associated with allergic airway disorders . we therefore investigated the potential involvement of ifn-2 in allergic airway diseases in the present study . to our surprise , information on the ifn-2 expressing cells is very limited . it was found that ifn-2 expressed in tracheobronchial tissue cells from the patients with copd . dc express moderate quantity of ifn-2 when using lipopolysaccharide ( lps ) as the maturation stimulus , and vitiligo patient skin and/or peripheral blood mononuclear cells express ifn-2 mrna . in order to understand the role of ifn-2 , we examined the cell origins of ifn-2 in the present study . the aim of the study is to investigate the expression of ifn-2 in peripheral blood of allergic airway disorders , its correlation with cytokines and tryptase , and its potential cell location . we found that the levels of ifn-2 were elevated in the plasma of ar and ar + as and that several cell types express ifn-2 . trypsin , leupeptin , collagenase ( type i ) , hyaluronidase ( type i ) , rabbit anti - human ifn-2 antibody , and bovine serum albumin ( bsa , fraction v ) were purchased from sigma aldrich ( st . louis , mo , usa ) . the sequences of the active and reverse peptides were par-2 , trans - cinnamoyl - leu - ile - gly - arg - leu - orn - amide ( tc - ligrlo - nh2 ) and trans - cinnamoyl - orn - leu - arg - gly - ile - leu - amide ( tc - olrgil - nh2 ) , sligkv - nh2 , and vkgils - nh2 ; par-2 antagonist peptide phe - ser - leu - leu - arg - asn - nh2 ( fsllrn - nh2 ) was synthesized in cl bio - scientific inc . dulbecco 's modified eagle 's medium ( dmem ) and fetal calf serum ( fcs ) were obtained from hyclone ( logan , ut , usa ) . human ifn-2 , il-4 , il-10 , and il-12 elisa kits were purchased from r&d systems ( minneapolis , mn ) . foxp3 fix / perm buffer set , rbc lysis buffer ( 10x ) , fitc - anti - human cd123 , percp - anti - human cd16 , percp - anti - human hla - dr , percp / cy5.5-anti - human cd25 , percp / cy5.5-anti - human il-17a , pe / cy7-anti - human cd8 , pe / cy7-anti - human cd14 , and pe / cy7 conjugated rat anti - human il-4 antibodies were purchased from biolegend ( san diego , ca , usa ) . fixation / permeabilization solution kit , fitc - anti - human cd4 , apc - anti - human cd19 , apc - anti - human ifn- , alexa fluor 647-anti - human foxp3 , and pe conjugated rat anti - mouse igm antibodies were purchased from bd pharmingen ( san jose , ca , usa ) . fitc or pe conjugated goat anti - rabbit igg antibody was purchased from santa cruz biotec ( santa cruz , ca , usa ) . biotinylated rabbit anti - human ifn-2 was purchased from bioss ( beijing , china ) . dab + substrate chromogen system and extravidin - peroxidase conjugate were purchased from chemicon international inc . recombinant human lung -tryptase was obtained from promega ( madison , wi , usa ) . exscript rt reagent kit and sybr premix ex taq ( perfect real time ) were obtained from takara biotechnology co. , ltd . oligonucleotide primers for real - time pcr were synthesized by invitrogen biotechnology co. ( shanghai , china ) . most of the general chemicals such as salts and buffer components were of analytical grade . a total of 33 allergic rhinitis ( ar ) , 26 asthma , 12 combined rhinitis with asthma ( ar + as ) , and 20 healthy control subjects ( hc ) were recruited in the study . the diagnosing criterion of asthma was conformed to the global initiative for asthma , and diagnosis for allergic rhinitis was based on allergic rhinitis and its impact on asthma ( aria ) . all patients were asked to stop antiallergy medication for at least 2 weeks prior to attending the study ( those who could not stop antiallergy drugs were excluded ) . the informed consent from each volunteer according to the declaration of helsinki and agreement with the ethical committee of the first affiliated hospital of liaoning medical university and general hospital of shenyang military area command were obtained . the general characteristics of the patients and control subjects were summarized in table 1 . peripheral venous blood sample ( 10 ml ) was collected from each patient or hc and was immediately processed to collect cells and plasma for analysis . specimens of human tissues for immunohistochemistry and flow cytometry analysis were collected from the department of pathology , the first affiliated hospital of liaoning medical university . the protocol for ethical use of human tissue in research was according to the declaration of helsinki ( 2000 ) and approved by the committees of the first affiliated hospital of liaoning medical university . to detect ifn-2 expression on leukocytes excluding t cells , the following antibodies were added to different testing tubes : ( 1 ) to detect ifn-2 expression in basophils : fitc - anti - human cd123 and percp - anti - human hla - dr ; ( 2 ) to detect ifn-2 expression in cd16 + polynucleated cells , cd16 polynucleated cells , and cd14 + cells and cd19 + cells : percp - anti - human cd16 , pe / cy7-anti - human cd14 , and apc - anti - human cd19 before 200 l of whole blood being added at room temperature for 15 min in dark . following ligation of red blood cells , white blood cells were fixed and permeabilized by using cytofix / cytoperm fixation / permeabilization kit according to the manufacturer 's instructions . following washing with bd washing buffer , the cell pellets were resuspended and rabbit anti - human ifn-2 followed by pe or fitc conjugated goat anti - rabbit igg antibodies were added at 4c for 30 min . finally , cells were resuspended in fluorescence - activated cell sorting- ( facs- ) flow solution and analyzed with facsverse flow cytometer ( bd biosciences , san jose , ca ) . data were analyzed with cellquest software ( bd immunocytometry systems ) . for detection of ifn-2 expression in t cells , peripheral blood mononucleated cells ( pbmc ) were isolated by using lymphoprep according to the manufacturer 's instruction . the following antibodies were then added to different testing tubes : ( 1 ) fitc - anti - human cd4 , percp / cy5.5-anti - human cd25 , pe / cy7-anti - human cd8 , and rabbit anti - human ifn-2 followed by alexa fluor 647-anti - human foxp3 and pe conjugated goat anti - rabbit igg antibodies to detect cd8 + t cells and regulatory t cells ( treg ) ; ( 2 ) fitc - anti - human cd4 , apc - anti - human ifn- , pe / cy7 conjugated rat anti - human il-4 , percp / cy5.5-anti - human il-17a , and rabbit anti - human ifn-2 followed by pe conjugated goat anti - rabbit igg antibodies to detect th1 , th2 , and th17 cells . cells were then incubated with each labeled monoclonal antibody including ( 1 ) pe / cy7 conjugated mouse anti - human tryptase , anti - human chymase antibody cc4 ( igm subtype ) , pe conjugated rat anti - mouse igm , rabbit anti - human ifn-2 , and fitc conjugated goat anti - rabbit igg antibodies to detect mast cells ; ( 2 ) pe / cy7-anti - human cd14 , apc - anti - human cd19 , rabbit anti - human ifn-2 , and fitc conjugated goat anti - rabbit igg antibodies to detect macrophages and b cells at 4c for 30 min in dark . after washing , the cell pellets were resuspended in facs - flow solution and analyzed with facsverse flow cytometer . tissues were fixed in carnoy 's fixative , dehydrated , and embedded in paraffin wax . sections ( 4 m ) were dewaxed , rehydrated , and incubated for 10 min with 0.5% h2o2 in methanol followed by 0.1% sodium azide for 10 min in order to inhibit endogenous peroxidase activity . pbs containing 5% bsa was added for 1 h and the same solution was employed as the diluent for the antibodies added subsequently . sequential sections of tonsil , lung , or nasal polyps were incubated with biotinylated rabbit anti - human ifn-2 for 2 h. after washing with pbst , extravidin - peroxidase conjugate was applied to sections for 1 h. staining was developed over 4 min by using dab chromogen system before being counterstained with mayer 's haematoxylin and mounted in aquamount . for each section , the number of positively stained cells was counted in at least 30 fields ( the area of each field equals 0.19 mm ) . the human lung carcinoma cell line a549 ( morphology : epithelial ) was obtained from the american type culture collection ( manassas , va , usa ) . cells were grown in dulbecco 's modified eagle 's medium ( dmem ) , supplemented with 10% ( v / v ) fetal calf serum ( fcs ) , 100 u / ml penicillin , and 100 g / ml streptomycin . cells were cultured in 75 cm tissue culture flasks ( falcon ) at 37c in a 5% ( v / v ) co2 , water - saturated atmosphere . for challenge experiments , cells were detached from culture flasks using trypsin , seeded into 12-well cell culture plates , and grown to about 80% confluence . the cells were then cultured with the serum - free basal medium for an additional 16 h before challenge . cells were exposed to tryptase ( 2 g / ml , 1 g / ml = 7.4 nm ) with or without its inhibitor leupeptin ( 3 g / ml ) , 100 m of sligkv - nh2 with or without par-2 antagonist fsllrn - nh2 ( 400 m ) and its reverse peptide vkgils - nh2 , and 100 m of tc - ligrlo - nh2 with or without par-2 antagonist fsllrn - nh2 ( 400 m ) and its reverse peptide tc - olrgil - nh2 , respectively . cells ( 1.5 10 per well ) were collected at 2 h or 6 h , centrifuged at 4c , and stored at 80c until use . the expression of ifn-2 mrna in a549 cells was determined by qpcr following the manufacture 's protocol . briefly , after synthesizing cdna from total rna by using superscript first strand synthesis system for rt - pcr and oligo - dt primers , real - time pcr was performed by using sybr premix ex taq kit on the abi prism 7700 sequence detection system ( perkin elmer applied systems , foster city , ca , usa ) . sequence - specific standard curves were generated using 10-fold serial dilutions of plasmid dna , and the values for the initial concentrations of unknown samples were calculated by using the software ( version 1.7 ) provided with the abi 7700 system . the primers for ifn-2 were forward : 5-caccctgcaccatatcctct-3 , reverse : 5-ggagggtcagacacacaggt-3 and for -actin were forward : 5-agagctacgagctgcctgac-3 , reverse : 5-agcactgtgttggcgtacag-3. levels of tryptase , il-4 , il-10 , il-12 , and ifn-2 in the plasma of ar , asthma , ar + as , and hc were measured by using elisa kits according to the manufacturer 's instructions . data were expressed as mean sem . where analysis of variance indicated significant differences between groups with anova , student 's t - test was applied . data for allergic patients are presented as scatter plot . where kruskal - wallis analysis indicated significant differences between groups , for the preplanned comparisons of interest , the paired mann - whitney u test was employed . in order to evaluate the potential role of ifn-2 in allergic airway disorders , the most direct evidence is to examine the changes of its levels in clinical specimen . we therefore examined the levels of ifn-2 in the plasma and its cellular location in blood of the patients with ar and asthma . the results showed that the levels of ifn-2 were elevated by 17.9% and 14.2% in the plasma of ar and combined rhinitis with asthma ( ar + as ) , but not of asthma ( figure 1(a ) ) . the plasma levels of tryptase were increased by 34.7% and 38.3% in the patients with ar and asthma , but not ar + as ( figure 1(b ) ) . the plasma levels of il-4 were increased by 21.1% in the patients with asthma but decreased by 55.3% and 26.3% in ar and ar + as ( figure 1(c ) ) . the plasma levels of il-10 ( figure 1(d ) ) and il-12 ( figure 1(e ) ) were decreased by 29.8% and 100% in the patients with ar , by 54.3% and 100% in the patients with asthma , and by 100% and 100% in the patients with ar + as , respectively . there were positive correlation between ifn-2 and tryptase and negative correlation between ifn-2 and il-10 in the plasma of ar . similarly , plasma ifn-2 positively correlates with tryptase , and il-10 positively correlates with il-12 in asthma ( table 2 ) . in order to identify the potential sources of ifn-2 the results showed that ifn-2 was predominately expressed in the cd16 + ( representing neutrophils ) ( figure 2(a)(f ) ) and cd14 + cells ( representing monocytes ) ( figure 2(a)(e ) ) and weakly expressed in cd19 + ( representing b cells ) ( figure 2(a)(a ) ) , cd8 + cells ( representing cytotoxic t cells ) ( figure 2(a)(b ) ) , and basophils ( figure 2(a)(g ) ) . cd4 + t cells ( figure 2(a)(d ) ) and cd16 polynucleated cells ( representing eosinophils ) ( figure 2(a)(c ) ) seemed not to express ifn-2 in hc ( figure 2(b ) ) . however ifn-2 expression was upregulated by 43.5% and 49.1% in ar , by 125% and 42.3% in asthma , and by 99% and 72.8% in ar + as in cytotoxic t cells and eosinophils but downregulated by 57% and 76.3% in ar , by 86.4% and 81.6% in asthma , and by 58.1% and 37.2% in ar + as in monocytes and neutrophils , respectively ( figure 2(b ) ) . in order to further investigate the potential source of ifn-2 , we examined the expression of ifn-2 in cells of various tissue origins by using immunohistochemical staining technique . the results showed that ifn-2 clearly expresses in glandular epithelial cells and some large cells ( most likely mast cells or macrophages ) in tonsillar tissue ( figure 3(b ) ) and in some large cells in lung tissue ( figure 3(d ) ) and nasal polyps ( figure 3(f ) ) as compared with the negative control tissues ( figures 3(a ) , 3(c ) , and 3(f ) ) . to confirm the immunohistochemical staining observations , we examined ifn-2 expression in dispersed human tonsil and lung mast cells , b cells , and macrophages by flow cytometry analysis . the results showed that approximately 2.1% , 4.5% , and 7.0% dispersed tonsil cells are ifn-2 + mct mast cells , mctc mast cells , and macrophages . however , 2.5% , 3.3% , 0.44% , and 0.14% dispersed cells are ifn-2 + mct mast cells , mctc mast cells , macrophages , and b cells ( figure 4 ) . positive correlation of ifn-2 with tryptase implicated that the increased level of ifn-2 in the plasma of patients with ar and ar + as may be elicited by mast cell tryptase . to confirm this anticipation , we examined the effect of tryptase and agonist peptides of par-2 on ifn-2 mrna expression in a549 cells . it was found that the expression of ifn-2 mrna over baseline control was increased by approximately 1.4- and 0.5-fold when the cells were incubated with tryptase at 2 g / ml for 2 and 6 h ( figure 5 ) . similarly , sligkv - nh2 and tc - ligrlo - nh2 induced approximately 1.4- and 0.9-fold increase in expression of ifn-2 mrna over baseline control , respectively , when they were incubated with a549 cells for 2 h ( figure 5 ) . at 6 h following incubation with sligkv - nh2 and tc - ligrlo - nh2 , the expression of ifn-2 mrna was enhanced by approximately 0.6- and 1.0-fold , respectively ( figure 5 ) . the reverse peptides vkgils - nh2 and tc - olrgil - nh2 showed little effect on the expression of ifn-2 mrna in a549 cells following 2 and 6 h incubation periods ( figure 5 ) . since fsllrn - nh2 and leupeptin were able to inhibit tryptase induced upregulation of expression of ifn-2 mrna and fsllrn - nh2 suppressed sligkv - nh2 and tc - ligrlo - nh2 induced upregulation of ifn-2 mrna expression ( figure 5 ) , the action of tryptase is likely to be mediated by par-2 and requires its enzymatic activity . we have demonstrated for the first time that the levels of ifn-2 are elevated in plasma of the patients with ar and ar + as , but not with asthma , which provides the first hard evidence for proving that ifn-2 may participate in adoptive immune response such as allergic airway reactions . the recent reports that the expression level of ifn-2 mrna was significantly increased during naturally occurring respiratory viral infections in children with asthma and that ifn-2 was capable of exacerbating a t - cell - mediated autoimmune disease may support our observation . it is difficult to evaluate the role of ifn-2 in allergic airway inflammation at this stage as we do not know if the increased serum level of ifn-2 is a causative or resulting factor in the pathogenesis of the allergic airway disorders . our observation that elevated ifn-2 levels were positively correlated to tryptase level in the plasma of ar suggests that these two compounds are likely released from the same source . since tryptase is a relatively selective marker of mast cell degranulation and the most abundant secretory product from mast cells , it is likely that ifn-2 is also released from mast cells upon degranulation . indeed , we have demonstrated in the present study that large numbers of tonsil and lung mct and mctc subtypes of mast cells express ifn-2 , confirming that mast cells are the major source of ifn-2 . our previous report that ifn-1 ( il-29 ) highly expressed in mast cells may support our current observation . however , unlike tryptase acting as a potent proinflammatory mediator which is capable of provoking microvascular leakage in the skin of guinea pigs , stimulating the release of histamine from dispersed human tonsil mast cells , and inducing accumulation of eosinophils and neutrophil in the peritoneum of mice , ifn-2 appears to act as a suppressor of allergic airway diseases . for example , ifn-2 can modulate lung dc function to promote th1 immune skewing and suppress allergic airway disease . since the information on the role of ifn-2 in allergy is very limited , the study that treatment with ifn-2 completely halts and reverses the development of collagen - induced arthritis , dramatically reduces numbers of proinflammatory il-17-producing th17 and t cells in the joints and inguinal lymph nodes , and restricts recruitment of il-1b - expressing neutrophils may support the anticipation that ifn-2 may play an inhibitory role in allergic airway diseases . since a large population of macrophages express ifn-2 , it is likely one of major sources of ifn-2 , considering huge numbers of macrophages in lung and tonsil . epithelial cells could be another source of ifn-2 as tonsil glandular epithelial cells express ifn-2 , and a549 cells express ifn-2 mrna . our observation that tryptase induced upregulation of expression of ifn-2 mrna in a549 cells is mediated by par-2 and requires tryptase enzymatic activity implicates that tryptase may provoke ifn-2 production in lung epithelial cells through activation of par-2 , and released ifn-2 could contribute to the elevated plasma level of ifn-2 in allergic airway disorders . is known of the relationship between pars and ifn-s , our previous report that the actions of thrombin on a549 cells are most likely carried out through hydrolytic cleavage of n - terminal of par-1 may help to understand our observation above . we have also observed the declined plasma levels of il-10 and il-12 in the allergic patients . since the correlation between il-12 and il-10 levels in serum has been reported in the patients with atopic dermatitis , and diminished il-12 levels were previously found in the serum of allergic patients , our observation may further suggest that reduced il-10 and il-12 production may contribute to the pathogenesis of the airway allergic disorders . the negative correlation between ifn-2 and il-10 in the plasma of ar suggested they are not likely to be released from same sources , which means that if mast cells are major source of ifn-2 , they should not be the major source for il-10 in ar . in order to identify the potential source of increased ifn-2 our data showed that ifn-2 expression was downregulated in ar , in asthma , and in ar + as in monocytes and neutrophils . since neutrophils and monocytes are predominant ifn-2-expressing cells in blood of hc , the decreased expression of ifn-2 in these 2 cell types could contribute to diminished level of ifn-2 in the plasma of asthma , even though ifn-2 expression appeared to be upregulated in blood cytotoxic t cells and eosinophils in asthma as cytotoxic t cells only weakly express and eosinophils do not express ifn-2 in hc . downregulation of expression of ifn-2 in peripheral blood monocytes and neutrophils of ar and ar + as seemed to conflict with the observation of increased level of ifn-2 in the plasma of ar and ar + as , which suggests that there must be some other sources to generate large amount of ifn-2 apart from blood leukocytes . moreover since helper t cells including regulatory t cells do not express ifn-2 , they are one of the major sources of il-10 , which may at least partially explain the negative correlation between ifn-2 and il-10 in the plasma of ar . in conclusion , the elevated levels of ifn-2 in the plasma of ar and ar + as and positive correlations of plasma ifn-2 with tryptase in ar and asthma indicate that ifn-2 is likely to contribute to the pathogenesis of allergic airway disorders . mast cells , macrophages , and epithelial cells in human tonsil and lung tissues express ifn-2 , and upregulated ifn-2 expression was observed in cd8 + t cells and eosinophils of allergic airway disorders indicate that they are the potential sources of ifn-2 . sodium nitrate ( nano2 ) is one of important environmental toxicants and poses important health risks . most countries use nano2 as food additive as a source of color and flavor preservation in meat products and this chemical can also be found in water resources . consumption of nano2 at low levels for long time caused histopathologic changes , nitrosative tissue damage , and lipid peroxidation in liver and kidney , as well as inducing chromosomal aberrations , decreased immune system , increased cancer colon risk , hypoxia , enlargement of the carotid body , and a vasodilation due to the activity with haemoglobin in the blood to form methaemoglobin , which has a much higher ( up to 20 times ) affinity for oxygen [ 4 , 5 ] . acute intraperitoneal treatment of wistar rats with nano2 ( at the dose of 50 mg / kg bw ) influences the blood rheological properties and erythrocyte hematometric indices . as very little data are available on the effort to cure the intoxication of nano2 , the aim of this work was to evaluate the curative effect of chlorophyll from sauropus androgynus ( l ) merr as antioxidant to cure its toxic effect in inducing oxidative stress . dietary chlorophyll can be found in fresh fruits and vegetables as chlorophyll a and chlorophyll b , thermally processed fruits and vegetables as metal - free pheophytins and pyropheophytins , and thermally processed green vegetables as zn - pheophytins and zn - pyropheophytins . chlorophyll in form of underutilized greens in fresh vegetables , supplements , liquid solutions , extracts , or tablets can be used effectively as healthy and beneficial nutrient supplement . antioxidant activity is one of the beneficial effects of chlorophylls to prevent oxidative dna damage and lipid peroxidation both by reducing reactive oxygen species ( ros ) and chelating metal ions [ 811 ] . chlorophylls can act as a hydrogen donor to break the chain reaction , due to the porphyrin in its chemical structure . sauropus androgynus ( l ) merr was identified as potentially rich sources of chlorophyll [ 13 , 14 ] . the antioxidant activities of the dark green leaves of s. androgynous were reported to have biologically nutritive value . among others , they have antidiabetic activity in diabetic mice induced by alloxan , reduce fever , increase breastmilk production , and prevent hoarse voice ; have antidyslipidemic activity and prevent the cardiovascular disorder in wistar male rats induced with fat - rich diet ; affect the growth performances , resist diseases , and enhance nonspecific immune responses in grouper diets . the previous study reported that antioxidant activity of chlorophyll from s. androgynous leaves is able to decrease schistocytes percentage and malondialdehyde ( mda ) level and also increase the level of haemoglobin ( hb ) and ferritin in female mice induced by nano2 . this work may provide new information for toxicological testing to validate the safety and traditional uses of these plants . cu - chlorophyllin reported has a higher antioxidant activity than that of natural chlorophylls due to the presence of the chelated metal in the porphyrin ring . the antioxidant activities of the cu - chelated compounds were found to be much higher than those of natural chlorophylls and of mg - free derivatives . this study also compared the curative effect of natural chlorophyll in s. androgynus leaves compared to cu - chlorophyllin antioxidant activity in female rats induced subacute sodium nitrite ( nano2 ) . haematological blood assays and the level of mda , ferritin , and transferrin in blood serum were analysed as curative effect indicator of chlorophyll , while the histopathologic view of liver and kidney tissues was used to evaluate its toxicity . fresh leaves of s. androgynus ( l ) merr were collected from the inhabitant park in penggaron lor village , genuk , semarang , central java , indonesia . cu - chlorophyllin from k - liquid was obtained from a drug store in semarang , central java , indonesia . a given amount ( 200 g ) of fresh leaves was cleaned and was thereafter blended with acetone : methanol ( 7 : 3 , v / v ) using an electric blender until the pigment was removed . the organic phase material that resulted was added steadily with na2so4 anhydrate to ensure no water in the extract . the solvent removal and drying process of chlorophyll used rotary evaporator and n2 gas . a suspension of 0.016 mg of chlorophyll from s. androgynus ( l ) merr leaf extract ( csa ) was dissolved in 1 ml distilled water , used as a dose 1 of csa , while for making 1/2 dose csa 0.008 mg / ml was diluted from 1 dose csa . thirty female wistar albino rats ( 150200 g ) were obtained from the faculty of pharmacy , universitas gadjah mada , yogyakarta , indonesia . rats were maintained in the animal house of faculty of medicine , universitas islam sultan agung in standard hard bottom polypropylene cages at 23c 2c , 12 : 12 h light / dark cycle and free access to laboratory chow and tap water throughout the study . ethical clearance was obtained from the ethics committee of the faculty of medicine , universitas islam sultan agung , indonesia , with document number 150/v/2015/komisi bioetika . nano2 was administered 50 mg / kg body weight ( dissolved in 1 ml distilled water ) , which refers to rate of ld50 on rats . nano2 as much as 1 ml / day was given intraperitoneally to the rats for 10 days for treated animals ( groups ii , iii , iv , v , and vi ) . the control rats ( group i ) were treated with the same volume of distilled water . during the following 14 days , the rats from groups iii and iv were given orally dose 1/2 ( 0.008 mg / ml ) and dose 1 ( 0.016 mg / ml ) of csa , respectively , whereas the rats from groups v and vi were given cu - chlorophyllin from k - liquid with dose 1/2 ( 0.008 mg / ml ) and dose 1 ( 0.016 mg / ml ) , respectively . cu - chlorophyllin was dissolved in distilled water based on the instruction in the packing . the solution of chlorophyll and cu - chlorophyllin , those given to rat , were 3 ml / head / day according to the conversion of adult man doses . blood samples were collected from the animals via the periorbital sinus 24 h after the last treatment . about 3 ml of the blood the clotted blood samples were centrifuged at 3000 rpm for 10 min to obtain the serum , which was used for biochemical analyses . another 2 ml of the blood samples was collected into heparinized tubes that were used for haematological assays . the packed cell volume ( pcv ) red blood cell ( rbc ) counts were determined using the new improved neubauer hemocytometer . schistocytes are detected in the peripheral blood smear stained using wedge procedures and observed by microscopy in 100x every 1000 erythrocytes . commercially available kits were used according to the respective manufacturer 's protocol for the measurement of mda , ferritin , and transferrin . mda level was measured using thio barbituric acid reactive substance ( tbars ) test with 532 nm wavelength spectrophotometer , whilst ferritin level was measured using enzyme linked immunosorbent assay ( elisa ) method . all the animals were then sacrificed by anesthetizing with diethyl ether 24 h after the last treatment . the organ tissues were processed and embedded in paraffin wax and sections were made of about 46 m . after staining with haematoxylin and eosin , slides were examined under the microscope ( olympus , japan ) for histopathological changes and photographed . the histopathologic parameters for liver were scored as follows : ( 1 ) showing no changes or normal , ( 2 ) parenchymatous degeneration , ( 3 ) hydropic degeneration , and ( 4 ) necrosis , while the scoring for proximal tubule epithelium was as follows : ( 1 ) lesion less than 25% indicating no change , ( 2 ) lesion 25%<50% , ( 3 ) lesion 50%<75% , and ( 4 ) lesion more than 75% showing severe changes . results are reported as mean values sem and statistically analysed by one - way anova test with 95% significance level . if the data characteristics did not allow for the one - way anova test to be conducted , then the kruskal - wallis test became the alternative . the hb in induced nano2 rats group was significantly lower ( p < 0.05 ) than that of rats in control groups . it indicated that the subacute administration of nano2 can cause anaemia which was indicated by the decrease of hb level , pcv percentage , increase the rbc concentration , and schistocytes percentage . changes in blood parameters and immune response are the direct toxic effects due to high dose administration of nano2 . pvc indicates the proportion of whole blood occupied by the rbc and depends on the hb level in rbc . the percentages of schistocytes in nano2-induced rats group were significantly higher than control group which proved that nano2 is able to damage the resulting fragmented rbcs called as schistocytes . the schistocytes may have different forms such as triangular , helmet , or comma shaped of broken or fragmented erythrocytes . nano2 affects the haematological and hemorheological parameters in mature rats [ 5 , 24 ] . the group that was treated with one - dose chlorophyll from s. androgynous showed significant difference in hb level , rbc concentration when compared with the nano2 group ( p < 0.05 ) , but no significant difference with the cu - chlorophyllin in the same dose and control group . this indicated that the natural chlorophyll from s. androgynous leave is as effective as cu - chlorophyllin to cure the oxidative stress caused by nano2 induction . the haematological parameters as pcv , hb , and schistocytes in in the lower dose group of s. androgynous chlorophyll and cu - chlorophyllin were not significantly different ( p > 0.05 ) compared to those in the nano2 group , which indicated that lower dose of chlorophyll was not more effective at ameliorating the anaemia symptoms induced by nano2 . nano2 is one of methaemoglobin - forming drugs that may exacerbate oxidative toxicity under certain chronic or acute hemolytic settings . the increasing of hb level showed that nano2 induced erythrocyte methemoglobinemia by increasing reactive oxygen species ( ros ) . the fusion of the heme moiety of hemoglobin released from red blood cells into endothelium could provide catalytically active iron to the vasculature . ferritin as a cytoprotection against free radicals in vitro will be increased in the increasing of ros . the mda level increased significantly in nano2 induction group , whereas transferrin and ferritin level were reduced significantly with exposure to nano2 when compared with control . nitrate is reduced to nitrite and nitrite is converted to nitrosonium ions which in turn reacts with amines and amides to form nitrosamines and nitrosamines , respectively . n - nitrosamines have the ability to induce rapid oxidative stress and then cause lipid peroxidation ; therefore it disturbs the cellular homoeostasis . the serum mda level is one of the molecules used as indicator of lipid peroxidation to estimate oxidative stress . present study showed that rats who received nano2 for 10 days showed increasing of the mda serum significantly compared to control group . this result is in accordance with the previous studies that elevated mda level in nano2-given mice and elevated levels of serum mda in rats given nano2 . results from this study revealed that treatment with chlorophyll of s. androgynus restored mda levels after nano2 treatments to normal values . the mda level in group ii which was not treated by the chlorophyll remained high ( see table 2 ) . the decreasing of mda levels in group iii till group vi after the nano2 treatments is the results of the antioxidant activity of chlorophyll of s. androgynus and cu - chlorophyllin . the same effects were observed for serum ferritin and transferrin . decreased serum transferrin and ferritin levels could be a result of damage , particularly protein oxidation by reactive oxygen species generated by nano2 toxicity . there was a decrease ( p < 0.05 ) in serum transferrin and ferritin in the nano2-induced rat followed by therapy with s. androgynus chlorophyll and cu - chlorophyllin group compared with the control group . the histopathologic parameters for liver and proximal tubule epithelium of kidney were not significantly different among groups ( table 3 ) , although there were increasing score of hepatocyte and proximal tubule epithelium damage in nano2-induced group . histopathology of hepatocyte of rat liver in control group showed normal or no change with score 1 , while the hepatocyte in nano2 induced group showed hydropic degeneration ( figure 1 ) . the hepatocyte and proximal tubule kidney in group treated with dose 1 cu - chlorophyllin showed the highest score of hepatocyte histopathologic view even not significantly different from the control . there was founded necrosis cell in proximal tubule kidney of rats treated by cu - chlorophyllin ( figure 2 ) . the histology of kidney in the group cured with dose 1 of chlorophyll showed that the lesion cell was less than 50% . in the present study , intake of nano2 for 10 days this result was in accordance with roth and kate smith that rats given nano2 via drinking water at 13 g / l during pregnancy and lactation showed decreasing of erythropoietic development , changing in some histopathological such as cytoplasmic vacuolization of centrilobular hepatocytes , and decreased hematopoiesis in bone marrow and spleen . have detected hydropic cellular degeneration in liver and tubular degeneration in kidney in 20 mg / kg / day nano2-induced mice for 8 months . the histopathological changes are mediated by oxidative stress produced by the action of the metabolic pathways generated against the toxic compounds . ( 2005 ) reported that cu - chlorophyllin tested by -carotene bleaching method and the stable radical 2,2-diphenyl-1-picrylhydrazyl ( dpph ) scavenging assay methods presented a higher antioxidant activity than that of natural chlorophylls , showing the importance of the nature of the chelated metal in the porphyrin ring . the mechanism of antioxidant activity displayed by the natural chlorophyll derivatives does not seem to be based on the ability to donate hydrogen but maybe , the protection of linoleic acid against oxidation , and/or preventing decomposition of hydroperoxides . this is suggestive of hepatocytes protection from antioxidant activity from chlorophyll of s. androgynus against nano2-induced damages . the histopathologic parameters for liver showed nano2-induced toxicity and ameliorative potentials of chlorophyll of s. androgynus on nano2-induced oxidative stress . chlorophyllin copper ( cu(ii)-chlorophyllin ) is one of derivatives of chlorophyll that reported increased peripheral leukocyte count and improving symptoms of dizziness and fatigue in individuals with leukopenia . chlorophyll is converted to mg - free pheophytin derivatives during digestion and then transported to peripheral tissues . chlorophylls degraded rapidly to pheophytins in response to the high acidity of the gastric phase . in the past , chlorophyll has been used to treat gastrointestinal problems , anaemia , and cancer . fahey et al . reported that chlorophyll can improve the function of essential detoxification pathways . chlorophyllin was 410-fold more potent as a phase 2 enzyme inducer than chlorophyll , since it has other detoxification properties because it is much more water - soluble than chlorophyll . . showed that sasa senanensis rehder leaf extract containing fe(ii)-chlorophyllin demonstrated superoxide anion and hydroxyl radical - scavenging activity five times higher than a similar product containing cu(ii)-chlorophyllin and comparable to a product containing cu(ii)-chlorophyllin , ginseng , and pine leaf extracts . therefore , the data obtained from this study further proved the usefulness of chlorophyll of s. androgynus as a food supplement that may be recommended to cure humans and animals from nano2 toxicity . the present study has demonstrated the curative effect of chlorophyll solution from s. androgynus leaves on haematological , serum biochemical , and histological parameters , altered by sodium nitrate exposure in female wistar rats . this amelioration may be partly due to the antioxidant activity of chlorophyll from s. androgynus leaves that possess remarkable potential to cure the oxidative stress caused by sodium nitrate . further assessment of molecular evaluations of biological activities of chlorophyll from s. androgynus needs to be studied .

this study investigated the expression levels of interferon- ( ifn- ) 2 in peripheral blood and tissues . the results showed that the levels of ifn-2 were elevated by 17.9% and 14.2% in the plasma of allergic rhinitis ( ar ) and combined rhinitis with asthma ( ar + as ) , which was positively correlated with the level of tryptase but negatively correlated with the level of il-10 . ifn-2 was predominately expressed in the cd16 + cells and cd14 + cells in healthy control subjects ( hc ) but upregulated only in cd8 + cells of ar and in eosinophils of asthma . it was observed that approximately 6.6% and 7.0% dispersed tonsil cells and 5.8% and 0.44% dispersed lung cells are ifn-2 + mast cells and macrophages . moreover , tryptase and agonist peptides of par-2 induced enhanced ifn-2 mrna expression in a549 cells . in conclusion , the elevated levels of ifn-2 in the plasma of ar and ar + as indicate that ifn-2 is likely to contribute to the pathogenesis of allergic airway disorders . the potential origins of the elevated plasma ifn-2 include mast cells , macrophages , and epithelial cells in tissues , neutrophils , monocytes , cd8 + t cells , and eosinophils in peripheral blood . development of ifn-2 related therapy may help to treat or prevent allergic airway disorders . sodium nitrate ( nano2 ) widely used as food additive for coloring and preserving meat has been reported to induce oxidative stress and cause histopathologic changes , nitrosative tissue damage , and lipid peroxidation in liver and kidney . therefore , the present study compared the curative effect of chlorophyll from sauropus androgynus ( l ) merr and cu - chlorophyllin as antioxidant in nano2-induced female wistar rats based on haematological , serum biochemical , and histological evaluation . thirty male wistar rats were randomly assigned into six groups of five rats each . nano2 were given at a subacute dose of 50 mg / kg bw intraperitoneally for 10 days . chlorophyll from s. androgynus and cu - chlorophyllin from k - liquid were given in the following 14 days at the two doses : 0,016 mg / ml and 0.008 mg / ml . nano2 exposure resulted in significant reductions ( p < 0.05 ) in values of packed cell volume ( pcv ) , haemoglobin ( hb ) concentration and red blood cell ( rbc ) count , transferrin , and ferritin and elevation in malondialdehyde ( mda ) level and schistocytes percentage with insignificant reductions in serum albumin and transferrin levels . histology of kidney and liver were changed insignificantly ( p > 0.05 ) to normal values . chlorophyll from s. androgynus and cu - chlorophyllin possess antioxidant potentials to protect against toxicities induced by sodium nitrate .

placental polyp is a somewhat pedunculated remnant of chorionic tissue retained in the uterine cavity for an indefinite time . it may result in abnormal uterine bleeding and slightly elevated detectable titers of serum -human chorionic gonadotropin ( hcg ) . these pedunculated masses of villi are often found within days to weeks following abortion or delivery of a term placenta . since trophoblastic neoplasms especially placental site trophoblastic tumor may have similar symptoms and signs , it is important to consider placental polyp in differential diagnosis in such situations . a 34-year - old g4l3ab1 woman came with abnormal uterine bleeding since her last normal vaginal delivery 3 months ago . serum -hcg level was slightly elevated ranging from 86 to 103 iu / ml during diagnostic investigations . ultrasonography revealed enlarged uterus with an echolucent intracavitary uterine mass measuring 73 mm 55 mm 24 mm . computerized topography confirmed the presence of the mass and showed no abnormality in thorax . clinical , laboratory , and imaging findings raised the suspicion of gestational trophoblastic tumors especially those arising from intermediate trophoblastic cells . macroscopically , the uterus showed slight global enlargement resulting from the presence of a polypoid mass within the endometrial cavity . the cut surface was diffusely red with some fine streaks of a gray colored tissue . it was attached to the uterine wall in the fundal region without any macroscopic permeation into the myometrium [ figure 1 ] . microscopic study showed largely necrotic villi in a network of fibrin deposition [ figures 2 and 3 ] . a large polypoid mass with smooth outer surface has completely filled the endometrial cavity necrotic chorionic villi are seen in the background of fibrin deposition ( 40 ) nuclear debris are seen in the stroma of necrotic chorionic villi ( 400 ) placental polyp is a fragment of retained placental tissue in the uterus that has undergone neovascularization after resolution of gestation . chronic uterine inversion due to placental polyp has also been reported . a case of placental these pedunculated masses of villi are often found within days to weeks following abortion or delivery of a term placenta . rarely , they persist for months or even years after pregnancy . abnormal uterine bleeding due to placental polyp has been attributed to preserved villi , clusters of destructive villi , and isolated viable cotyledons . preservation of the brush border of syncytiotrophoblastic cells and the presence of placental phosphatase maintain the anticoagulative properties of villi . thromboplastic properties of the preserved villi play an important role in the pathogenesis of uterine bleeding when necrotic villi with epithelial remnants are prevalent . computed tomographic angiography is also useful in diagnosis and management of placental polyp with neovascularization . magnetic resonance imaging may also be used in diagnosis and follow - up of placental polyps . . a hypervascular placental polyp may lead to severe hemorrhage that requires blood transfusions , interventional radiology procedures , hysteroscopic resection , and even hysterectomy to control bleeding . evaluation of neovascularization by multimodal imaging is potentially useful in management of placental polyp in women who wish to preserve fertility . successful treatment with the use of iliac artery occlusion catheters and concomitant hysteroscopic resection has been reported . intraoperative injection of prostaglandin f2 followed by hysteroscopic resection has been successful in management of these cases . serum hcg fell to undetectable level following surgery . although the patient had completed her family and did not have any desire to preserve her fertility , a proper preoperative diagnosis with accurate interpretation of imaging findings and satisfactory curettage would have prevented hysterectomy in this patient . placental polyp should be considered in any case of parous woman with unexplained abnormal uterine bleeding and slightly elevated serum hcg level . this does not exclude the possibility of the presence of a placental polyp as the source of abnormal bleeding . all authors have contributed in designing and preparation of the first draft of the manuscript . they have read and approved the content of the manuscript and confirmed the accuracy or integrity of any part of the work . halogen bonding has emerged in recent years as an effective alternative to hydrogen bonding in directing the formation of self - assembling architectures , with fruitful applications in many chemical and biological systems . halogen bonding is an electrostatically driven noncovalent interaction between a halogen atom in one molecule and a lone - pair ( n ) or -electron donor in another . the electron - withdrawing effect of groups covalently bound to a halogen atom depletes the electron density in the n orbital to yield an electropositive -hole . halogen bonding serves as an ideal cohesive force in self - assembling systems due to its linear directionality , tunable bonding strength , and stability in hydrophobic environments . in addition to attractive intermolecular forces , self - assembling processes may be aided via cooperative effects , i.e. , nonadditive enhancement from polarization and charge transfer . the existence of cooperativity in halogen bonding has been identified in previous crystallographic and theoretical studies covering molecular complexes with varied structures and strengths . the present computational study expands upon previous efforts to quantitatively examine cooperative effects and optimal motifs for linear and multiply halogen bonded systems and to consider their potential in the construction of self - assembling architectures . by performing density functional theory ( dft ) calculations , enhanced understanding is sought on the nature of cooperativity through geometrical , energetic , and natural bonding orbital ( nbo ) analyses . factors are considered that could influence interaction strength such as polarization , secondary interactions , and spacing between halogen bonds . recently , modeling of halogen bonding interactions in supramolecular systems has been facilitated in force fields by representing the -hole as a partial positive point charge attached to the halogen atom . carter et al . also redesigned a force field for halogen bonds by including angular dependency into the standard lennard - jones potential . in the present work , model systems are considered and then extended to construct cylindrical complexes analogous to carbon nanotubes ( cnts ) . the initial focus was on 4-bromopyridine and 1-bromo-1h - imidazole as prototypical building blocks for construction of larger , self - assembling systems . the first issue was to evaluate the structures , interaction strengths , and cooperativity for linear oligomers , namely , ( 4-bromopyridine)n ( 1 ) and ( 1-bromo-1h - imidazole)n ( 2 ) with n = 16 , as shown in figure 1 for n = 4 . as described in computational methods , dft calculations were carried at the m06 - 2x/6 - 31+g(d , p)-lanl2dzdp - pp level with counterpoise corrections using geometries optimized with the b97x functional and the same basis set . as reported in table 1 , the calculated average halogen - bond lengths decline and binding energies strengthen steadily with growing oligomer size . the average halogen - bond strength increases for the 4-bromopyridine case from 2.94 to 3.08 kcal / mol for n = 26 . the interactions and cooperative effects for the imidazole case are notably stronger , advancing from 8.31 kcal / mol for the dimer to 10.30 kcal / mol for the hexamer . the halogen bonds are also significantly shorter with nbr separations near 2.5 for 2 versus 3.0 for 1 . the much stronger halogen bonds for 2 are due to the nitrogen atom being covalently bound to bromine , which simultaneously enhances electron withdrawal from bromine by induction and electron donation to the lewis basic nitrogen by resonance . most of the linear chains of both compounds exhibit nonuniform sequential arrangements , with shorter halogen - bond distances in the middle and longer ones toward the ends of the chains . this geometric characteristic has also been found in hydrogen - bonded linear chains . as also shown in table 1 ( column 3 ) , including zero - point energy ( zpe ) corrections optimized geometries of linear chains for tetramers of 4-bromopyridine ( 1 ) and 1-bromo-1h - imidazole ( 2 ) . percentage change compared to the dimer . the energy difference between the optimized complex and separated monomers including zero - point energy corrections . cooperative effects are apparent in the increasing average strengths of the halogen bonds . further quantification comes from evaluating the average cooperative energy , ecoop , according to eq 1 for oligomers of sizes greater than 2 . as shown in table 1 , the negative values for ecoop confirm the existence of cooperative effects in all cases , and they increase in magnitude as the chains grow.1 a many - body analysis was also carried out using the standard partition scheme of hankins et al . for the trimers and tetramers . this requires calculations for each constituent n - mer to yield the total interaction energy as a sum of contributions from two- , three- , and four - body interactions . the results in table 2 indicate that while the two - body interactions make the dominant contributions to the binding energies , the three - body terms are substantial and increase significantly in going from the trimers to tetramers . however , the four - body terms are negligible , as found in a previous many - body analysis of hydrogen - bonded hcn chains . to further characterize the cooperative effects , the investigations considered the changes in dipole moment and intermolecular orbital interactions via nbo analysis . the average dipole moment per molecule ( ) and the cooperative dipole moment ( coop , defined analogously to the cooperative energy , increase for addition of each monomer ( table 3 ) . the calculated average intermolecular charge transfer ( qct ) and delocalization energies ( e ) associated with the lone pair on the nitrogen atoms ( nn ) and antibonding orbitals of the c br bonds ( *c br ) are also found to increase with the size of the oligomers in table 3 . moreover , figure 2 illustrates that the average binding energy correlates linearly with average dipole moment ( correlation coefficient = 0.998 and 0.994 ) and transferred charge ( correlation coefficient = 0.954 and 0.981 ) . overall , the present results support the consensus view that polarization is a major source of the cooperativity in linear halogen - bonded systems . correlations of the average halogen bonding energy ( exb , kcal / mol ) with the average dipole moment ( debyes ) and average amount of charge transferred ( e ) for linear oligomers of ( 4-bromopyridine)26 on the left and ( 1-bromo-1h - imidazole)26 on the right . dipole moments in debyes ; transferred charges in e ; energies in kilocalories per mole . while previous studies have focused on the cooperativity in linear systems , the cooperativity in multiply halogen bonded systems was also explored here for a variety of haloazines . the goals are to identify the factors that influence halogen - bond strengths and to determine optimal halogen - bonding motifs for potential use in self - assembly of large systems . as shown in figure 3 and table 4 , results were obtained for the singly , doubly , and triply halogen bonded complexes 3a3c . these cases illustrate alternation of the donor and acceptor sites at the interface . the average length of the halogen bonds increases slightly along this series , which likely reflects the unfavorable hh repulsions across the interfaces for 3b and 3c . nevertheless , the average strength increases from 2.94 to 3.39 kcal / mol . . 10 kcal / mol , which is stronger by 1 kcal / mol than for the tetramer of 1 , also featuring three halogen bonds . thus , the cooperative effect is somewhat greater for extension of the halogen bonding system laterally rather than in a linear , head - to - tail manner . halogen bond lengths in the crystal structures from ref ( 32 ) . including zero - point energy corrections . related systems that incorporate extensive two - dimensional networks of nitrogen chlorine halogen bonds have been reported in crystallographic studies . in the present work , two of the azaaromatic chlorides were revisited : cyanuric trichloride ( 3d ) and 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine ( 3e ) . the dimer and heptamer of 3d and the dimer and tetramer for 3e were optimized . as shown in table 4 , the dft results for the halogen - bond lengths for the oligomers of 3d and 3e are in excellent agreement with the findings from the x - ray crystallography . however , compared to the bromoazines , the dft results reveal that the halogen bonds for 3d and 3e are significantly weaker with average strengths of 1.51.9 kcal / mol . similarly , in our prior study of the complexes of bromobenzene and chlorobenzene with pyridine , binding energies of 2.7 and 1.9 kcal / mol were obtained . though the halogen bonds for 3d and 3e are weak , they clearly contribute to the packing and self - assembly reflected in the crystal structures . stronger halogen bonding interactions can be expected to lead to more robust self - assembling materials . continuing in figure 3 , 3f and 3 g remove or add a benzene spacer to 3b . the binding energy for 3f is significantly weaker at 3.37 kcal / mol owing to the repulsion between the central bromine atoms ; the monomers shift laterally such that the c brn angles are 164 rather than the optimal 180 for halogen bonds . however , the additional spacer in 3 g has little impact yielding an interaction energy nearly the same as that for 3b . complexes 3h and 3i are isomeric with 3f and 3 g ; however , both donors and both acceptors are now on the same side of the interface . for hydrogen - bonded systems , this arrangement can lead to stronger binding owing to the favorable secondary electrostatic interactions . however , in 3h the repulsion between the bromines causes them to bend away from each other with c c br angles of 125 and c brn angles of 162 , which leads to weak binding of only 3.55 kcal / mol . the problem is relieved for 3i , though its binding energy of 5.60 kcal / mol is about 0.7 kcal / mol weaker than for 3 g . there are small structural differences in this case ; the halogen bonds are more linear for 3 g than 3i with c finally , 3j and 3k explore replacement of the benzene rings in 3b and 3c with cyclohexyl spacers . the nbr halogen bond lengths are constant at 3.093.10 ; however , the partially saturated systems exhibit stronger attraction with binding energies of 7.16 and 11.34 kcal / mol for 3j and 3k . the increased attraction can be attributed to reduced hh repulsion across the interfaces . for the unsaturated cases such as 3b , 3c , and 3 g , the shortest interannular hh contacts are 2.82.9 , while they are 3.43.5 for the partially saturated 3j and 3k . thus , for the motifs in figure 3 , the strongest average halogen bond strengths occur when the donor and acceptor sites alternate on each side of the interface and when there is a spacer ring between the 4-bromopyridine subunits as in 3b and 3j . significant cooperative effects are apparent with the average nbr halogen bond strength climbing from 2.94 kcal / mol in the reference dimer 3a to 3.39 kcal / mol in 3c and to 3.78 kcal / mol in 3k . this analysis assigns all of the net interaction to the halogen bonds , which is an oversimplification since there are at least varying steric effects associated with the hh interactions at the interface . however , the results establish that multiply halogen bonded interfaces can be constructed where the net interaction is more attractive than from the sum of the individual halogen bonds . in view of the possible influence of secondary electrostatic interactions on complexation , six additional dimers related to 3b were considered ( figure 4 and table 5 ) . each complex formally has two nbr halogen bonds ; however , the interface has been modified by the arrangement of the donors and acceptors or by modification of the central ring as benzene , pyridine , or pyrazine . compound 4a is the isomer of 3b with both donors on one side of the interface and both acceptors on the other . as with 3 g and 3i , the preference is for the alternating arrangement in 3b over the parallel one in 4a by nearly 1 kcal / mol . the introduction of the interfacial nn repulsion does weaken the binding to 5.28 kcal / mol . this replaces the nn repulsion with an nh attraction , and the binding is enhanced to 6.40 kcal / mol . six alternative doubly halogen bonded complexes with their computed binding energies ebind ( kcal / mol ) from m06 - 2x/6 - 31+g(d , p)-lanl2dzdp - pp calculations . primary interactions are indicated with solid lines and secondary ones are shown with dashed lines . distances in ngstroms ; energies in kilocalories per mole . including zero - point energy corrections . complexes 4d and 4e are the analogues of 3b with the pyrazine and pyridine spacers ; they are also isomers of 4b and 4c with the donor and acceptor arrangement returned to alternating . these complexes both show some enhancement of the binding energy to 6.79 and 7.03 kcal / mol . for 4d the interannular nn repulsion is diminished by a lateral shift such that the nbr halogen bonds become somewhat bifurcated . the outer c brn angle is 173 in 4d , while the inner one is 148 , and the central nn distance is 5.0 . in 4e , compound 4f is the final possibility in this series ; it is the isomer of 4c with the three nitrogen atoms on one edge . the binding weakens to 6.06 kcal / mol , which suggests that the basicity of the nitrogens in 4f is lessened by their proximity . n bonds induces slight concave curvature to the nitrogen - containing edges in 4f that splays out the opposite bromine - containing edges . in turn , this adversely affects the linearity of the halogen bonds in 4f with c brn angles of 171 vs 178 in 4c . these results establish that 3b , 3j , 4d , and 4e represent the motifs with the most attractive interactions . they feature 4-bromopyridine units with edges of alternating donor and acceptor sites separated by a monocyclic spacer . they can all be extended to potentially yield sheetlike structures in the solid state and a foundation for construction of other self - assembling materials . the possible utility of the 3b motif for constructing tubelike structures that could self - assemble was considered . cylindrical belts were generated with alternating 4-bromopyridine and benzene rings and alternating up / down arrangement of the bromopyridines . dimers of the belts with six ( 5a ) , eight ( 5b ) , and ten ( 5c ) repeats can form six , eight , and ten halogen bonds at each interface ( figure 5 ) . the dimers were modeled with dft and molecular mechanics calculations . for the latter , the fixed - charge force field , opls / cm1ax , was used as well as the version that includes polarization via induced dipoles on non - hydrogen atoms , opls / cm1axp . both force fields incorporate partial positive charges ( x - sites ) to represent the -holes on chlorine , bromine , and iodine atoms . the energetic results for 5a , 5b , and 5c are summarized in table 6 . in view of the increasing system sizes , two alternatives for the dft method and basis sets were needed . the geometry optimizations for 5b and 5c used the smaller 6 - 31g(d)-lanl2dzdp - pp basis set with blyp , and the isolated monomer geometry was taken to be the same as in the optimized dimer . supramolecular tubes can be assembled from cylindrical belts using six ( 5a ) or ten ( 5c ) halogen bonds at each interface . using m06 - 2x//b97x with the 6 - 31+g(d , p)-lanl2dzdp - pp basis set . using m06 - 2x//b97 - 1 with the 6 - 31g(d , p)-lanl2dzdp - pp basis set . using m06l/6 - 31g(d , p)-lanl2dzdp - pp//blyp/6 - 31g(d)-lanl2dzdp - pp . for 3b and 3c as references , the binding energies from the force field calculations are found to be in near perfect agreement with the m06 - 2x results . this is not unexpected since opls / cm1ax was parametrized to reproduce halogen - bonding results from high - end ab initio calculations , specifically , mp2(full)/aug - cc - pvdz(-pp ) . 5c where the best accord is found between the nonpolarizable opls / cm1ax and dft results . as noted previously , the cm1a charges include the intramolecular electronic polarization for the monomers , and the intermolecular polarization is generally only essential for modeling interactions with small ions . notably , along the 5a5c series the binding intensifies from 20 to 27 to 35 kcal / mol . there is also a gradual increase in the average halogen bond strength from 3.24 to 3.46 kcal / mol according to the dft calculations . this cooperative effect is not mirrored in the force field results which yield the same average halogen bond strength for the two larger systems . the strong binding for 5a5c indicates that the halogen bonding motifs in 3b , 3j , 4d , and 4e could be used as the cohesive elements to build a wide range of interesting supramolecular systems and nanomaterials . furthermore , the excellent accord between the dft and opls / cm1ax results supports the use of opls / cm1ax as a computationally efficient means for modeling such systems . the present study addressed halogen binding strengths and cooperativity in linear and multiply halogen bonded systems . for the linear chains 1 and 2 , the cooperativity is largely a polarization effect , as supported by the close correlations of the cooperative dipole moments and amounts of charge transferred with the interaction energies . for multiply halogen bonded complexes such as 3a3c , the average halogen bond strength was also found to increase with increasing numbers of halogen bonds . four motifs , 3b , 3j , 4d , and 4e , emerged as having particularly strong halogen bonding and symmetries that allow their elaboration to yield large periodic systems . this notion was further explored with the demonstration that molecular belts based on 3b could be constructed that should self - assemble into nanotubes . furthermore , the opls / cm1ax force field was confirmed to accurately predict the interaction energies for halogen - bonded systems as large as the belt dimers 5a5c . the 6 - 31+g(d , p ) basis set was employed for all atoms with the exception of bromine , for which the lanl2dzdp - pp basis set with pseudopotentials was used . vibrational frequency calculations were carried out at the same level to confirm that the optimized structures were true minima . binding energies were obtained by single - point energy calculations with the m06 - 2x or m06l functional using the same basis set as the geometry optimization , and with basis set superposition error ( bsse ) removed via the boys bernardi counterpoise ( cp ) method . the accuracy of the results from such computations has been supported by numerous benchmark studies . natural bond orbital ( nbo ) analysis was performed with gaussian 09 following standard procedures . geometry optimizations were also performed using molecular mechanics as implemented in the boss 4.9 software . the opls / cm1ax force field , which features 1.14*cm1a partial atomic charges with extra point charges ( x - sites ) representing the -hole for halogen atoms , was used . the effects of inclusion of intermolecular polarization with inducible dipoles were considered with the opls / cm1axp force field . the polarizabilities , , for carbon , nitrogen , and bromine were assigned as 1.0 , 1.5 , and 2.0 .

placental polyp is retained placental tissue within the endometrial cavity , which forms a nidus for inflammation and bleeding . there are very few reported cases of the clinical placental polyp . here , we report a case of 34-year - old g4l3ab1 woman with the chief complaint of intermittent vaginal bleeding since her last normal vaginal delivery 3 months ago . serum human chorionic gonadotropin ( hcg ) titer was slightly elevated . a polypoid mass was detected within the endometrial cavity by imaging studies . history of the patient , mass lesion within the endometrial cavity and slightly elevated serum hcg titer raised the suspicion of trophoblastic neoplasms . endometrial curettage yielded unsatisfactory specimen containing only fibrin deposition and was followed by total hysterectomy . the uterus showed slight global enlargement resulting from the presence of a polypoid mass within the endometrial cavity . the red - colored mass had a smooth outer surface and fragile consistency without any permeation into the myometrium . pathology reported it as the placental polyp . although very rare , placental polyp should be kept in mind as one of the reasons of abnormal uterine bleeding in parous women . definite diagnosis is made by pathology examination . halogen bonding , due to its directionality and tunable strength , is being increasingly utilized in self - assembling materials and crystal engineering . using density functional theory ( dft ) and molecular mechanics ( opls / cm1ax ) calculations , multiply halogen bonded complexes of brominated imidazole and pyridine are investigated along with their potential in construction of self - assembling architectures . dimers with 110 halogen bonds are considered and reveal maximal binding energies of 336 kcal / mol . cooperative ( nonadditive ) effects are found in complexes that extend both along and perpendicular to the halogen bonding axes , with interaction energies depending on polarization , secondary interactions , and ring spacers . four structural motifs were identified to yield optimal halogen bonding . for the largest systems , the excellent agreement found between the dft and opls / cm1ax results supports the utility of the latter approach for analysis and design of self - assembling supramolecular structures .

in recent years , the development of an integrated nanotherapeutic system which can allow for an online imaging and deliver targeted therapy has been the subject of interest in clinical oncology . this emerging nanotechnology has allowed the manipulation of nanoparticles ( nps ) to assume multiple functionalities.14 interestingly in that view , the use of multifunctional quantum dots ( qds ) as fluorescence imaging contrast agents as compared with the traditional organic fluorophores has significantly enhanced both in vitro and in vivo detection and online assessment of cancer tumors in animal models.4,5 the preference of qds as optical imaging contrast agents as compared with the latter is largely due to its intrinsic ability to resist photobleaching.6,7 fluorescence semiconductors offer advantages in that , as they have a broad and tunable absorption spectrum extending from the ultraviolet ( uv ) toward the visible ( uv - vis ) region.8 semiconductor nanocrystals such as bulk zinc sulfate ( zns ) emits high fluorescence with a color that depends on the particle size but regrettably suffers from self - quenching effects . the doping of zns with other impurities such as mn and cd are reported to significantly reduce this intrinsic quenching behavior and at the same time enhances the fluorescence efficiency of the qds toward the cut - off wavelength of the visible region.8 interestingly , the similarity in the ionic radii and valence state of the mn and those of zn ions allowed for an easy substitution to take place in the host zns crystals.9 the mn ion in this regard serves as the luminescence center by providing recombination sites for the electrons and holes as charge carriers . the resultant transfer of electrons and hole charges into the electronic level of mn ions allow for the emission of a characteristic orange red fluorescence following t1a1 transition of the mn ion.1012 by the application of appropriate bioconjugation , the orange red fluorescence emission from the doped mn in the zns nanocrystals can conveniently replace the conventional organic fluorophore in molecular imaging and quantitative cellular studies.6,7,9 several researchers have explored novel approaches for the synthesis of water - soluble qds through the selection of appropriate stabilizers such as 3-mercaptopropionic acid ( mpa ) , mercaptoacetic acid , thioglycolic acid ( tga ) , polysaccharide copolymers , etc.4,13 however , some researchers observed that the quantum yield obtained from qds stabilized by thiol - groups is usually low.1416 furthermore , the thiol - qd bond is also reported not to be very stable and precipitates readily on being dialyzed against buffer solution and water with time.17,18 in addition to this finding , mpa , one of the common stabilizers used for the synthesis of water - soluble qds , is a volatile liquid with strong odor and is reported to be carcinogenic in nature and environmentally unfriendly . thus , the use and application of mpa as water - soluble stabilizer is not entirely green chemistry approach.19 in this regard , chitosan ( cs ) , a natural copolymer of n - acetylglucosamine and d - glucosamine has drawn greater attention as stabilizer of choice for the encapsulation of qds . cs is highly biocompatible , nontoxic and environmentally friendly and helps to render qds as water soluble.4,17 in addition to these qualities , the large functional backbone of cs can equally serves as a viable platform for the encapsulation of drugs and other suitable ligands.9,20 this unique signature of cs in addition to being a stabilizer and drug delivery vehicle , also protects the conjugated qds and reduces the possibility of nonspecific protein opsonization.17,21 from a biomedical point of view , the mn - doped zns ( mn : zns ) conjugated cs nanocarrier can be made to achieve active or selective targeting and imaging of diseases by attaching suitable ligands with strong binding affinity toward the largely expressed receptors such as proteins , vitamins , monoclonal antibodies , aptamers , and peptides.22,23 these among many have been reported in the development of a number of theranostic applications that included targeted drug delivery using receptor - mediated pathways for the detection of cancerous tumors or following protein protein interactions toward the detection of proteins in blood assays.2426 the active targeting has attracted considerable interest due to its target specificity unlike the passive targeting , which takes advantage of leaky vasculature found in cancerous cells through enhanced permeability and retention effects to transport the drug.27 under these processes , there exists the possibility of the required dosage being lost on transit before reaching the diseased site.28 in that view , folic acid ( fa ) , a nontoxic low - weight vitamin is found to have strong binding affinity toward folate receptors ( frs ) largely secreted by cancerous cells as compared with the normal cells.21 though fa was successfully conjugated directly to the surface of metallic nanoparticles ( mnps ) such as qds,29 some researchers observed that this often resulted in the oxidation of the surface and possible changes in the optical chemistry of the mnps and the unprotected or naked mnps were exposed to the scavenging effects of blood matrix species.21 thus , in our work the fa was first conjugated to cs and following this conjugation , the qds were then encapsulated . it is therefore hypothesized that the functionalization of mn : zns conjugated cs with fa and encapsulation with an anticancer drug helps to achieve triple purpose : 1 ) enabling specific targeting of folate - expressed cancer cells ; 2 ) as a vehicle for stabilizing and transporting drugs to the tumor - targeted sites ; and 3 ) the highly intense fluorescent emission from mn can be used for noninvasive imaging of the cancer cells . here , we present an aqueous , room temperature synthesis of fluorescence mn : zns qds loaded onto cs biopolymer and conjugated to fa for specific targeting of fr - overexpressed cancer cells following modified protocols reported elsewhere.4 in this study , we intentionally avoided the use of lengthy spacer groups so as to retain the structural integrity of our materials as much as possible , and also avoided the rigor of chemical modification that may introduce possible structural defects and/or alteration of the electronic states of the core material.2831 it has been demonstrated that the fa conveniently binds to the surface of metals via noncovalent interactions31,32 and similar method of interaction with cs were also reported.33 in this particular instance , the conjugation of the fa and cs is initiated due to the strong electrostatic interaction of the cationic amino groups of cs with the anionic carboxylate group of fa ; the result shows a loading efficiency of approximately 30.51.2 wt.%.33 despite the successes recorded following noncovalent method of conjugation of fa to other biomolecules or mnps,2934 several work reported a superior performance following covalent methods of interactions.4,35,36 however , the greatest challenge is meeting the requirements of specific conjugation chemistry that retains qds biochemical activity as much as possible . despite the superiority of covalent coupling of fa to qds by means of 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide - based chemistry , some researchers reported that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide has short half - life in water and results in poor coupling efficiency . although the addition of n - hydroxysuccinimide may have improved the efficiency,36 it is considered to follow lengthy pathways that may probably modify the biochemical integrity of the qds / nps . in the same vein , other researchers reported that the proteins readily lost their structural integrity upon adsorption onto monolayer - protected metal nps ( or qds ) following covalent interactions , leading to instability and subsequent denaturation.37 it is also investigated that the drug whose active site is covalently bound to the surface of nps are observed not to undergo controlled drug release readily as compared with drugs encapsulated by following noncovalent interactions.38 therefore for controlled and targeted delivery , formulation of nanocarriers with noncovalent interactions may be the probable method of preserving the active ingredients and structural integrity of the materials as much as possible . hence , the nonchemical methods adopted in this work follows simple ultrasonication , centrifugation , and controlled ph.2933 low - molecular - weight cs ( 75%85% degree of deacetylation ) was purchased from sigma - aldrich ( st louis , mo , usa ) , while the other chemicals such as ethanol ( 99.5% ) was purchased from fisher scientific ( selangor , malaysia ) and acetic acid ( 99.85% ) from benson lab chemicals ( selangor , malaysia ) . zinc acetate dehydrate ( zn ( oac)22h2o ) 99.5% , fa ( c19h9n7o6 , 98% ) , sodium sulfide ( na2sxh2o ) , manganese sulfate ( mnso4h2o , 99% ) , and sodium tripolyphosphate ( na5o10p3 , 59% ) were purchased from r&m marketing ( essex , uk ) . chitosan stabilized mndoped zns ( facs - mn : zns ) qds , the three sequential steps involved are demonstrated schematically in figure 1 . the first step involves the formation of stable fa and cs suspension and for that , we prepared 5 ml ( at 4 mg / ml concentration ) of water - soluble cs by protonating the nh2 group with 1% acetic acid39 under stirring while adjusting the ph to 5.4 using 0.1 m naoh . further , 2 ml of 1 mg / ml fa in distilled water was prepared separately and stirred for 1 hour to form fa solution and the resulting solution was then added to the cs solution dropwise under constant stirring for 2 hours . now 2 ml of 1 mg / ml tripolyphosphate was added as a cross - linker drop by drop to the mixture and stirred for 8 hours to form the stable suspension of facs . the second step involves the synthesis of highly luminescent mn : zns qds following simple modified wet method as described by mathew et al.4 briefly , 1.5 ml of mnso4 ( varying at.% ) was prepared and added dropwise to 10 ml zn(oac)2 ( 0.1 m ) under ultrasonication and stirred well by bubbling with n2 gas for 15 minutes . to the resulting mixture , 10 ml of na2s ( 0.1 m ) was added drop by drop under constant stirring to form a white precipitate in the process while maintaining the ph to ~5.4 . the doping processes were observed for the emission of orange fluorescence under handheld uv lamp . in order to suppress unwanted relaxation phenomenon from dominating the energy pathways , the obtained precursor was immediately exposed to microwave irradiation for 1 minute and further stirred for 1 hour.4,10 the conjugation of facs to the mn : zns qds was carried in the third step and for that , the mn : zns qds as prepared above was added dropwise to the 10 ml facs solution under ultrasonication , allowed to sonicate for 10 minutes and stirred for another 1 hour . following the stirring , the centrifugation and washing procedure was repeated thrice and finally , the wet pellet was collected and air dried for 2 days to obtain the powder of facs - mn : zns nanocomposite . for the characterization of facs - mn : zns composite , different instruments were applied at various stages of the analysis ; for example , the particle size and surface morphology was determined using high - resolution transmission electron microscopy ( hrtem ) collected on tecnai g2 f20 ( fei company , hillsboro , or , usa ) and the samples were prepared by dispersing the particles in distilled water and drop casted onto a carbon - coated copper grid . similarly , gold sputtered samples were prepared and collected on a sample holder attached to a constructive aluminum foil using carbon adhesive for further morphology studies using field emission scanning electron microscopy ( fesem ) on jeol jsm-7600f ( jeol , tokyo , japan ) . elemental analysis of the samples were simultaneously recorded using the same instrument attached to energy dispersive analysis x - ray ( edax ) analyzer by focusing the electron beam onto the particles and the spectrums were obtained through the random capturing of micrographs . x - ray photoelectron spectroscopy ( xps ) measurements were carried out at a pressure of 10 mbar , using phi quantera ii ( ulvac - phi , inc , chigasaki , japan ) instrument operating with an alk monochromatic source . fourier transform infrared ( ftir ) analysis was conducted on the samples over 4,500500 cm wavenumber region at a resolution of 8 cm with 1,024 scan using spectrum 100 ( perkinelmer , waltham , ma , usa ) . for that , the transparent pellets of the samples were prepared by grinding the material with dry powder of potassium bromide . for the x - ray fluorescence ( xrf ) analysis , approximately 1 g of the sample was mixed with 6 g h3bo3 and pressed under 10 tons of pressure and was used to further determine the elemental composition using xrf - energy dispersive x - ray spectroscopy ( edx ) 720 , wavelength - dispersive spectrometer ( shimadzu , kyoto , japan ) equipped with an array of five analyzing crystals fitted with rh x - ray tube target . the dynamic light scattering ( dls ) analyses were conducted to measure the average particle size and distribution of the particles in solution phase with the help of malvern nano series , zetasizer instrument ( malvern instruments , malvern , uk ) . the dispersed sample in deionized water was collected in a cuvette and triplicate measurement was taken as mean standard deviation ( sd ) . the crystallinity of the sample was determined using powder x - ray diffraction ( xrd ) instrument xrd 6000 ( shimadzu ) and the crystal structure was analyzed using nickel - filtered cuk radiation . the thermographic analysis ( tga ) was conducted on a tg 7 perkinelmer instrument at a heating rate of 10c min in nitrogen atmosphere and the weight losses up to 600c was recorded . the uv - vis spectroscopic analyses were carried out on perkinelmer lambda 35 spectrometer by dispersing the samples in deionized water in a wavelength range of 200500 nm . for the fluorescence measurements , shimadzu spectrofluorimeter , rf-5301pc was used and the samples were prepared by dispersing in deionized water and the measurements were conducted at slit width kept at 5 nm on both 328 nm excitation wavelength . the microscopic fluorescence imaging was conducted using confocal microscopy , olympus fv1000 ( singapore ) , with silicone immersion and oil- and water - immersion objectives equipped with gallium phosphine detector ( olympus ) . the cell viability was assessed based on the metabolic activity of the cells to reduce a soluble yellow tetrazolium salt ( mtt dye , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) to blue violet formazan crystals.40 briefly , two different human breast cancer cell lines , mcf-7 and mda - mb-231 and one healthy normal breast cell line , mcf-10 were maintained separately in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) and 1% antibiotics ( penicillin , 100 units / ml ; streptomycin , 100 g / ml ) in an incubator at 37c , 5% co2 , and in 95% humidity . approximately 10 cells / well were seeded onto 96-well plates and allowed to grow for 24 hours . at approximately 80% cell confluence level , the medium was replaced with fresh dmem containing 2% ( v / v ) fbs and varying concentrations ( 7500 g / ml ) of facs - mn : zns with phosphate - buffered saline ( pbs ) ( ph 7.4 ) and incubated for another 24 hours . following the incubation period , 20 l of mtt ( 5 mg / ml ) was added and incubated for another 34 hours . after the incubation , the solution was removed and 100 l dimethyl sulfoxide ( dmso ) was added ; the solubilized crystals were measured at 570 nm using the quant enzyme - linked immunosorbent assay plate reader ( biotek instruments , winooski , vt , usa ) . the percentage cell viability was then calculated with respect to the untreated control ( set at 100% ) and the values were taken as mean sd of three replicate experiments . for the cell imaging study , the respective cells were separately maintained in dmem supplemented with 10% ( v / v ) fbs and 1% antibiotics ( penicillin , 100 units / ml ; streptomycin , 100 g / ml ) in an incubator at 37c , 5% co2 , and in 95% humidity . the cells were washed with pbs ( ph 7.4 ) and seeded at two different densities 210 and 310 per well onto a cover glass inside of 24-well plate . further washing of cells were conducted using pbs and replaced with a fresh media containing the facs - mn : zns composite ( 100 g / ml ) and incubated for 4 hours at 37c . the cells were finally washed with pbs and fixed with 2% formaldehyde and mounted with mounting medium and imaged . the microscopic fluorescence imaging was conducted using confocal microscopy , olympus ( fluoview f1200 ) with silicone immersion and oil- and water - immersion objectives equipped with gallium phosphine detector . the fluorescence generated by the qds was captured using effective excitation 360 nm and 550640 nm emission filters . low - molecular - weight cs ( 75%85% degree of deacetylation ) was purchased from sigma - aldrich ( st louis , mo , usa ) , while the other chemicals such as ethanol ( 99.5% ) was purchased from fisher scientific ( selangor , malaysia ) and acetic acid ( 99.85% ) from benson lab chemicals ( selangor , malaysia ) . zinc acetate dehydrate ( zn ( oac)22h2o ) 99.5% , fa ( c19h9n7o6 , 98% ) , sodium sulfide ( na2sxh2o ) , manganese sulfate ( mnso4h2o , 99% ) , and sodium tripolyphosphate ( na5o10p3 , 59% ) were purchased from r&m marketing ( essex , uk ) . for the preparation of folic acid chitosan stabilized mndoped zns ( facs - mn : zns ) qds , the three sequential steps involved are demonstrated schematically in figure 1 . the first step involves the formation of stable fa and cs suspension and for that , we prepared 5 ml ( at 4 mg / ml concentration ) of water - soluble cs by protonating the nh2 group with 1% acetic acid39 under stirring while adjusting the ph to 5.4 using 0.1 m naoh . further , 2 ml of 1 mg / ml fa in distilled water was prepared separately and stirred for 1 hour to form fa solution and the resulting solution was then added to the cs solution dropwise under constant stirring for 2 hours . now 2 ml of 1 mg / ml tripolyphosphate was added as a cross - linker drop by drop to the mixture and stirred for 8 hours to form the stable suspension of facs . the second step involves the synthesis of highly luminescent mn : zns qds following simple modified wet method as described by mathew et al.4 briefly , 1.5 ml of mnso4 ( varying at.% ) was prepared and added dropwise to 10 ml zn(oac)2 ( 0.1 m ) under ultrasonication and stirred well by bubbling with n2 gas for 15 minutes . to the resulting mixture , 10 ml of na2s ( 0.1 m ) was added drop by drop under constant stirring to form a white precipitate in the process while maintaining the ph to ~5.4 . the doping processes were observed for the emission of orange fluorescence under handheld uv lamp . in order to suppress unwanted relaxation phenomenon from dominating the energy pathways , the obtained precursor was immediately exposed to microwave irradiation for 1 minute and further stirred for 1 hour.4,10 the conjugation of facs to the mn : zns qds was carried in the third step and for that , the mn : zns qds as prepared above was added dropwise to the 10 ml facs solution under ultrasonication , allowed to sonicate for 10 minutes and stirred for another 1 hour . following the stirring , the centrifugation and washing procedure was repeated thrice and finally , the wet pellet was collected and air dried for 2 days to obtain the powder of facs - mn : zns nanocomposite . for the characterization of facs - mn : zns composite , different instruments were applied at various stages of the analysis ; for example , the particle size and surface morphology was determined using high - resolution transmission electron microscopy ( hrtem ) collected on tecnai g2 f20 ( fei company , hillsboro , or , usa ) and the samples were prepared by dispersing the particles in distilled water and drop casted onto a carbon - coated copper grid . similarly , gold sputtered samples were prepared and collected on a sample holder attached to a constructive aluminum foil using carbon adhesive for further morphology studies using field emission scanning electron microscopy ( fesem ) on jeol jsm-7600f ( jeol , tokyo , japan ) . elemental analysis of the samples were simultaneously recorded using the same instrument attached to energy dispersive analysis x - ray ( edax ) analyzer by focusing the electron beam onto the particles and the spectrums were obtained through the random capturing of micrographs . x - ray photoelectron spectroscopy ( xps ) measurements were carried out at a pressure of 10 mbar , using phi quantera ii ( ulvac - phi , inc , chigasaki , japan ) instrument operating with an alk monochromatic source . fourier transform infrared ( ftir ) analysis was conducted on the samples over 4,500500 cm wavenumber region at a resolution of 8 cm with 1,024 scan using spectrum 100 ( perkinelmer , waltham , ma , usa ) . for that , the transparent pellets of the samples were prepared by grinding the material with dry powder of potassium bromide . for the x - ray fluorescence ( xrf ) analysis , approximately 1 g of the sample was mixed with 6 g h3bo3 and pressed under 10 tons of pressure and was used to further determine the elemental composition using xrf - energy dispersive x - ray spectroscopy ( edx ) 720 , wavelength - dispersive spectrometer ( shimadzu , kyoto , japan ) equipped with an array of five analyzing crystals fitted with rh x - ray tube target . the dynamic light scattering ( dls ) analyses were conducted to measure the average particle size and distribution of the particles in solution phase with the help of malvern nano series , zetasizer instrument ( malvern instruments , malvern , uk ) . the dispersed sample in deionized water was collected in a cuvette and triplicate measurement was taken as mean standard deviation ( sd ) . the crystallinity of the sample was determined using powder x - ray diffraction ( xrd ) instrument xrd 6000 ( shimadzu ) and the crystal structure was analyzed using nickel - filtered cuk radiation . the thermographic analysis ( tga ) was conducted on a tg 7 perkinelmer instrument at a heating rate of 10c min in nitrogen atmosphere and the weight losses up to 600c was recorded . the uv - vis spectroscopic analyses were carried out on perkinelmer lambda 35 spectrometer by dispersing the samples in deionized water in a wavelength range of 200500 nm . for the fluorescence measurements , shimadzu spectrofluorimeter , rf-5301pc was used and the samples were prepared by dispersing in deionized water and the measurements were conducted at slit width kept at 5 nm on both 328 nm excitation wavelength . the microscopic fluorescence imaging was conducted using confocal microscopy , olympus fv1000 ( singapore ) , with silicone immersion and oil- and water - immersion objectives equipped with gallium phosphine detector ( olympus ) . the cell viability was assessed based on the metabolic activity of the cells to reduce a soluble yellow tetrazolium salt ( mtt dye , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) to blue violet formazan crystals.40 briefly , two different human breast cancer cell lines , mcf-7 and mda - mb-231 and one healthy normal breast cell line , mcf-10 were maintained separately in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) and 1% antibiotics ( penicillin , 100 units / ml ; streptomycin , 100 g / ml ) in an incubator at 37c , 5% co2 , and in 95% humidity . approximately 10 cells / well were seeded onto 96-well plates and allowed to grow for 24 hours . at approximately 80% cell confluence level , the medium was replaced with fresh dmem containing 2% ( v / v ) fbs and varying concentrations ( 7500 g / ml ) of facs - mn : zns with phosphate - buffered saline ( pbs ) ( ph 7.4 ) and incubated for another 24 hours . following the incubation period , 20 l of mtt ( 5 mg / ml ) was added and incubated for another 34 hours . after the incubation , the solution was removed and 100 l dimethyl sulfoxide ( dmso ) was added ; the solubilized crystals were measured at 570 nm using the quant enzyme - linked immunosorbent assay plate reader ( biotek instruments , winooski , vt , usa ) . the percentage cell viability was then calculated with respect to the untreated control ( set at 100% ) and the values were taken as mean sd of three replicate experiments . for the cell imaging study , the respective cells were separately maintained in dmem supplemented with 10% ( v / v ) fbs and 1% antibiotics ( penicillin , 100 units / ml ; streptomycin , 100 g / ml ) in an incubator at 37c , 5% co2 , and in 95% humidity . the cells were washed with pbs ( ph 7.4 ) and seeded at two different densities 210 and 310 per well onto a cover glass inside of 24-well plate . further washing of cells were conducted using pbs and replaced with a fresh media containing the facs - mn : zns composite ( 100 g / ml ) and incubated for 4 hours at 37c . the cells were finally washed with pbs and fixed with 2% formaldehyde and mounted with mounting medium and imaged . the microscopic fluorescence imaging was conducted using confocal microscopy , olympus ( fluoview f1200 ) with silicone immersion and oil- and water - immersion objectives equipped with gallium phosphine detector . the fluorescence generated by the qds was captured using effective excitation 360 nm and 550640 nm emission filters . in the present study , facs - mn : zns qds were prepared specifically for theranostic applications by combining the targeted drug delivery and imaging capabilities of fa and qds , respectively . the formed composite particles were thoroughly characterized for their size , morphology , crystalline behavior , thermal stability , etc , and also tested for cytobio - compatibility studies to suite as safe drug delivery vehicles . further , the in vitro fluorescence imaging studies on cancer and noncancer cells were conducted in order to establish the luminescent efficiency of the conjugated contrast agents for live cell imaging of cancerous tumors . the fesem and edax analysis of facs - mn : zns qds are shown in figure 2a and b and from the microscopic image , the particles seem to be agglomerated due to the over conjugation of qds with that of cs biopolymer ( figure 2a ) . also , from the edax spectrum shown in figure 2b , one can clearly see the presence of corresponding peaks related to specific elements of interest in the composite . the major peaks corresponding to zn and s are the indications for the presence of zn s matrix in the composite.41 interestingly , despite the small amount of mn impurities doped into the zns matrix , we were able to see a minor peak corresponding to mn in the final composite . as seen from the spectrum , the presence of au is from the au sputtered sample grid used in the fesem analysis.42 the hrtem image , particle size distribution from tem ( in powder phase ) , and dls analysis ( in solution phase ) are shown in figure 2c e , respectively . from the figure 2c , the particles are seem to be spherical and uniform but are fully agglomerated as similar to the result of fesem ( figure 2a ) . for the facs - mn : zns particles , an average particle size of 5.31.0 nm is observed from the tem analysis ( figure 2d ) , while ~200 nm hydrodynamic size is observed from the dls analysis ( figure 2e ) . the differences in particle sizes between the tem ( recorded in powder form ) and dls analysis ( recorded in solution form ) can be attributed to the swelling behavior of the cs polymer.43 the other possible explanation for the smaller particle size recorded by tem analysis is that , at lower acidic conditions ( ph ~56 ) , more amine and carboxylate groups from cs chain that are readily made available for bonding with the zn ions , thus acting as electron donors , which reduces the electrostatic repulsion and thereby favoring the stabilization of mn : zns nanocrystals to a more smaller and compact dimensions.44 thus from the cumulative analysis , the maximum particle size recorded for facs - mn : zns in both the solution phase ( dls , ~200 nm ) and powder form ( ~5.3 nm ) still falls within nanometer range . the elemental analysis of facs - mn : zns qds following xps and xrf analyses are shown in figure 3a e and table 1 , respectively . figure 3a shows the comparison of xps scanned surveys of facs - mn : zns composite and bare mn : zns qds of which both were prepared with 15 at.% of mn doping . one can see from the spectra that , only the peaks of zn , s , mn , o , and c are vividly appeared and when compared with bare mn : zns qds , a small peak corresponding to nitrogen ( n ) appearing only in the survey analysis of facs - mn : zns , providing the information about the conjugation of fa and cs molecules . further in figure 3b , the peak of zn 2p3/2 appeared at 1,021 ev for the bare mn : zns and 1,020 ev for facs - mn : zns confirming the existence of zn largely as zn linked to a sulfur atom . the binding energies of zn 2p3/2 and s 2p were only weakly affected by the conjugation of mn : zns with facs showing a shift of ~1 ev when conjugated with cs and fa . we also observed a very weak peak for mn 2p3/2 centered at 640 and 639 ev on conjugating with facs as represented in figure 3c . the observation of a weak peak can be due to the low concentration of mn element at the surface of the qds , a similar phenomenon was reported by some researchers following the doping of mn on zns nanocrystals.45,46 figure 3d shows the peak of s 2p3/2 , where the spectra for bare mn : zns qds has its peak centered at 162 ev , while the peak shifted to 160 ev on conjugation with facs . following the anchorage of mn : zns qds with cs and fa , the xps spectrum showed a signal of n 1s corresponding to the nitrogen atom from the amine groups of either the fa or cs ( figure 3e ) . the presence of elements in the composite was further substantiated from the xrf results shown in table 1 , approximately 80% and 13% corresponding to zn and s , respectively was recorded . however , it has been reported by some researchers that the actual concentration of mn ( at.% ) incorporated into the host zns is quiet less than the amount used during the synthesis . kole et al10 using electron probe microanalyzer reported that the optimum amount of mn concentration of 1.10 ( at.% ) corresponded to 40.0 ( molar % ) concentrations used during the synthesis . as a consequence of these observations , inductively coupled plasma - optical emission spectrometry analysis was used by some researchers to determine the actual concentration of mn . in their study , kole et al used 1 and 5 at.% of mn with respect to zn during the synthesis , but from the outcome of the analysis , the actual concentration of the mn incorporated into the final product was found to be 0.04 at.% for 1 at.% used and 0.18 at.% for 5 at.% used during the synthesis.10 these findings provide a proof for the observation of only trace amounts of mn recorded by edax , xps , and xrf analysis in our study . figure 4a c shows the powdered xrd pattern , thermal analysis , and ftir spectrums of mn : zns before and after conjugation with facs , respectively . the pattern in figure 4a indicates the formation of zns in a cubic blend ( sphalerite ) structure by the observation of peaks at 28.46 , 48.16 , and 55.28 corresponding to prominent diffraction at ( 111 ) , ( 220 ) , and ( 311 ) , respectively . the pattern shows no lattice distortion of the presence of mn impurities or any differentiated lattice structure with that of zn . one probable reason can be a result of the similarity in the oxidation state and ionic radii of mn with that of zn , in addition to the lower concentration of mn . however , a slight reduction in the peak intensity of facs - mn : zns is observed probably due to the complete coverage of mn : zns qds with that of cs and fa molecules . the thermal analysis studied for cs , mn : zns , and facs - mn : zns recorded in the temperature range of up to 600c ( figure 4b ) indicates the stability of mn : zns composite due to its conjugation with fa and cs . from the tga graph for pure cs , two curves are observed ; the observation of the first curve below 200c temperature can be attributed to the moisture loss from the sample , while the second curve around 230c is due to the decarboxylation of coo groups . also , the graph of mn : zns sample has some weight loss during 60c150c and this loss can be attributed to the evaporation of occluded moisture adsorbed at the surface of the particles . the particles displayed thermal stability after 300c up to 600c and finally left with around 50% of its initial weight . as similar to the other curves , the tga curve of facs - mn : zns shows a drop in weight from 50c to 120c due to the moisture loss and a slight depression during 150c450c either from the decarboxylation of the carboxylate groups or from loss of co2 . the observation of thermal stability in accordance with bare mn : zns in the temperature range of 450c600c is an indication for the persistence of mn and zn ions in the composite . furthermore , the thermal behavior of facs - mn : zns composite at 600c remaining with the highest weight of 70% as compared with the other two samples serves as a proof for the applicability of the concept of polymer composite formation to nanomaterials . the comparison of ftir spectra of facs - mn : zns composite with that of bare mn : zns , fa , and cs are shown in figure 4c . for the as - synthesized bare mn : zns , the characteristic absorption bands originated largely from zn s mn bonds only . also , the observation of a broad band in the bare mn : zns sample at 3,254 cm corresponds to the stretching vibration of hydroxyl ( o h ) group from the occluded water absorbed by the crystals ; similar vibration mode was also observed at 3,284 cm in the facs - mn : zns composite too . the two silent peaks at 2,358 and 2,166 cm can be due to zn s microstructure vibration and from co stretching vibrations , respectively . the zn s vibration peak ( at 2,358 cm ) observed in mn : zns is not pronounced in the spectra of facs - mn : zns and can be attributed to the interaction between the zn s and the carboxylate groups of fa or cs . the peaks at 1,406 and 1,544 cm in the spectra of mn : zns can be assigned to the symmetric and asymmetric stretching of coo , respectively , while the peak at 1,113 cm corresponds to the symmetric stretching vibration of zn s mn bond . this peak is however not visible in the spectra of facs - mn : zns probably due to the masking of c o c stretching vibrations of peak at 1,077 cm on cs s peak ( at 1,030 cm ) . this can also be the reason for the observation of a shifted peak at 1,019 cm in the spectra of mn : zns to 1,020 cm as seen in the spectra of facs - mn : zns . the small peaks at 906 and 1,183 cm in the spectra of facs - mn : zns can be from the aromatic c h out - of - plan bending of fa ( 908 cm ) and c both samples shows zns band and symmetric bending due to zn s or mn s vibrations at 656 and 609 cm for mn : zns and 667 and 614 cm in the spectra of facs - mn : zns , respectively . similarly , the peaks at 475 cm in the spectra of mn : zns and 465 cm for facs - mn : zns can be due to s s or mn o or s o interactions . the comparison of uv - vis absorption spectra of fa , bare mn : zns , and facs - mn : zns qds is shown in figure 5a and the tauc plot in figure 5b . as shown in the figure 5a , the absorption of fa is observed at 280 nm , while the bare mn : zns has absorption onset at 328 nm and facs - mn : zns at 318 nm . from the spectra , the optical absorption properties of the synthesized facs - mn : zns shows an absorption peak that is slightly red shifted from the absorption band edge of bulk zns qds ( at 334 nm ) owing to quantum confinement effects47 and following the doping of host zns with mn impurities.48 figure 5b shows the tauc plot of ( hv)2 versus ( hv ) from the uv - vis spectra and by extrapolation of the linear part of the curve to the energy axis based on equation 1 , the band gap energy of our sample ( eg ) facs - mn : zns qds was calculated to be 5.08 ev , a blue shift of approximately 1.4 ev from the band gap energy of bulk zns taking from literature value,10 hv = b(hveg)12(1)where is the absorption coefficient , hv is the photon energy , eg is the direct band gap energy , and b is a constant . the mn : zns qd s characteristic fluorescence behavior and its mechanism at various stages is fully demonstrated in figure 6a c . the figure 6a shows the comparison of fluorescence spectra of bare zns qds ( without mn doping ) and facs - mn : zns ( with mn doping ) . the fluorescence comparison of the two samples provides the information that the doping of zns qds with suitable impurity such as mn and independent of particle size can significantly enhance its luminescence properties . as seen from the spectra , the doping of zns with mn induces a red shift from the blue region at 450 nm , typical of undoped zns to more biofriendly visible region . the characteristic zns spectral shifted from the blue region toward the red region on doping with mn impurities and resulted in the emission of orange fluorescence at 600 nm . similarly , figure 6b shows what actually transpired following the doping chemistry , a change in color to orange when viewed under handheld uv lamp . from the jaboliski diagram shown in figure 6c , several mechanisms interplay to produce fluorescence emission in qds following the excitation of ground state electron to the excitonic state . the excited electrons either radiatively or nonradiatively relax and in the process , they recombine with the holes in the ground state with the emission of fluorescence light . in the case of zns as diagrammatically represented , the electron in the conduction band ( cb ) of zns lattice radiatively relaxes to the hole in the valence band ( vb ) passing through interstitial pathways of sulfur ( is ) and zn ( iz ) . the emission at 470 nm is due to the relaxation that occurs when the excited state electrons are trapped by sulfur vacancy donor levels.49,50 the mn ion has a d5 configuration , where the d - electron state plays a central role as the luminescence center by interacting strongly with the s p electronic state of the host zns in response to the electronic excitation.10 the resultant transfer of electrons and holes charges into the electronic level of mn ions allow the emission of characteristic orange red fluorescence following t1a1 transition of the mn ion.10 to further buttress the phenomenon surrounding the effect of doping of atoms to zns , several pathways are reported to take part during the excitation of mn in the host zns and the subsequent orange emission ( oe ) . as can be seen in figure 6c , three main possible pathways maybe responsible for the electron hole recombination that further leads to oe:50 in the first relaxation pathway , there exists the possibility that the electron in the cb of the zns lattice radiatively relaxes to the holes in the vb through is and iz ( ie , interstitial sites of sulfur and zinc ) . due to lattice strain induced by is and the large ionic radius of sulfur ion as compared with zn ions , the electrons initiated by is has small binding energy relative to zn ion.49 in the second relaxation pathway , it is possible that the blue emission can be observed at 475 nm from the relaxation that occurs when the electrons in the excited state are trapped by the sulfur vacancy donor levels.it is further considered that : a ) the excited electrons trapped by the sulfur vacancy level can nonradiatively relax to the t1 level of mn ; and b ) the electrons in the excited state may directly relax to the t1 level of mn by nonradiative transition . as previously mentioned , the electrons generated at t1 level of mn following either of the possible paths can recombine radiatively with the holes trapped in the ground state ( a1 ) of mn ions ; this accounts for the oe recorded in our study at ~600 nm . the fluorescence emission at the visible and far red is more intense than the blue emission regions . one possible reason can be due to the nonradiative transition of electrons from the cb to the t1 level of the mn ions is much faster than the electron captured by the sulfur vacancy level.51 in the first relaxation pathway , there exists the possibility that the electron in the cb of the zns lattice radiatively relaxes to the holes in the vb through is and iz ( ie , interstitial sites of sulfur and zinc ) . due to lattice strain induced by is and the large ionic radius of sulfur ion as compared with zn ions , the electrons initiated by is has small binding energy relative to zn ion.49 in the second relaxation pathway , it is possible that the blue emission can be observed at 475 nm from the relaxation that occurs when the electrons in the excited state are trapped by the sulfur vacancy donor levels . it is further considered that : a ) the excited electrons trapped by the sulfur vacancy level can nonradiatively relax to the t1 level of mn ; and b ) the electrons in the excited state may directly relax to the t1 level of mn by nonradiative transition . as previously mentioned , the electrons generated at t1 level of mn following either of the possible paths can recombine radiatively with the holes trapped in the ground state ( a1 ) of mn ions ; this accounts for the oe recorded in our study at ~600 nm . the fluorescence emission at the visible and far red is more intense than the blue emission regions . one possible reason can be due to the nonradiative transition of electrons from the cb to the t1 level of the mn ions is much faster than the electron captured by the sulfur vacancy level.51 the comparison of fluorescence behavior of facs - mn : zns qds as against the changes in the physical parameters such as uv light , ph , and dopant concentration are shown in figure 7a d . from the comparison of fluorescence spectra shown in figure 7a and b , there is a possibility of unwanted relaxation pathways recombining to produce the defect curve observed between 400 and 470 nm regions . this characteristic phenomenon was observed to be more pronounced on the synthesized samples in the absence of uv irradiation ( figure 7a ) and however observed to be milder on samples treated under uv irradiation ( figure 7b ) . the treatment of qds with uv irradiation being used by most researchers to enhance the luminescence center of mn - doped zns suppresses the activity of unwanted nonradiative recombination.1012 however , from the spectra , after treatment with uv irradiation , we expect a significant rise in the fluorescence intensity but on the contrary , a drop in intensity was recorded for facs - mn : zns . one possible conclusion is that , the polymer conjugated to the qds may induce a restriction of the rotational motion of dipole molecules and immobilization of the electron hole charge ions , thereby interfering with the surface chemistry.52,53 geszke et al44 reported two possible mechanisms as reason for the observed quenching effects : 1 ) the fa conjugated to the qds can interfere and trap part of the excitation light intensity driven by collisions between fa and qds ; and 2 ) fa readily conjugate to mn : zns through the carboxylate groups or through the nitrogen atoms . following this strong affinity toward each other , there exists the possibility that the highest occupied molecular orbital and lowest unoccupied molecular orbital of the fa overlap with the cbs of zn ( 4s ) initiating energy transfer from the mn : zns to the fa . in order to reduce the quenching effect as a result of the conjugation of the qds with fa , they observed and reported in their experiment that the luminescence intensity of qds was only weakly altered ( ~5% ) when the concentration at molar ratios of fa : qds was maintained near to 0.01 . the fluorescence intensity behavior measured as against the changes in external factors such as ph ( figure 7c ) and dopant ion concentration ( figure 7d ) indicates that they both have significant influence toward the fluorescence efficiency . as seen from the comparison of spectra ( figure 7c ) , the fluorescence intensity increases from ph 4 to 8 and it drops from ph 12 to a level below ph 8 . from the observed pattern , due to higher acidic environment at ph 4 , there is a possibility for the dissociation of carboxylate or amino groups present at the surface of qds and thereby distorting the coordination . also , the high alkaline environment at ph 12 favors the formation of metal oxides or hydroxides as a result of the presence of large number of oh ions . based on the observed pattern , fluorescence efficiency was recorded to be more stable at low acidic and low alkaline ph ( ie , ph around 68 ) . similarly , the changes in spectra from blue to visible region on doping of zns with mn were also observed to increase following an increase in the concentration of mn ( figure 7d ) . the comparison of cell viability and proliferation studies following the treatment of mn : zns and facs - mn : zns as against different cell lines are shown in figure 8a d . the mtt assay was performed on two breast cancer cell lines , mcf-7 ( figure 8a ) , mda - mb-231 ( figure 8b ) , and normal human breast cell line ( mcf-10 , figure 8c ) grown for 24 hours with medium containing bare mn : zns qds and facs - mn : zns with concentrations ranging from 7 to 500 g / ml . from the comparative analysis of the cells cultured without the composites as control , the results demonstrated that the qds did not exhibit discernable adverse effects in vitro in all the cell lines . the results show only a slight reduction in the cells viability in response to an increase in qds concentrations , especially pronounced from 62 to 500 g / ml on cells treated with facs - mn : zns as compared with those exposed to bare mn : zns qds . one possible reason for this slight reduction in cells viability for the cells treated with facs - mn : zns can be due to the enhanced cellular binding interactions between the conjugated fa - containing qds with the fr - expressed cancer cells . these relationships were further substantiated when comparing the mtt result of normal breast cell line ( mcf-10 ) with that of cancer breast cells ( mcf-7 and mda - mb-231 ) treated with facs - mn : zns ( figure 8d ) . from the result , the reduction in cells viability was observed to be more pronounced on cells expressing frs ( mcf-7 and mda - mb-231 ) as compared with the normal cells without frs ( mcf-10 ) , suggesting that the expression of frs on cancerous cells enhances the intracellular concentrations of qds conjugated with fa . the in vitro fluorescence imaging studies conducted on human breast cancer lines ( mcf-7 and mda - mb-231 ) that expressed frs and normal breast cells ( mcf-10 ) without frs are shown in figure 9a d . from the figure , it can be seen that the composite conjugated with fa demonstrated specific attachment by emitting out cellular fluorescence from the qds attached to the frs expressed on the cancer cells ( figure 9b - iii and c - iii ) . however , just the milder fluorescence effects were observed on cells administered with nonconjugated fa composites ( mn : zns , figure 9a - iii ) and conjugated fa composites ( figure 9d - iii ) . thus , the observed fluorescence property demonstrated by the fr - overexpressing cells ( figure 9b and c ) supported the hypothesis that facs - mn : zns qds can serve as suitable contrasting agents for the online targeted imaging of cancerous cells . in conclusion , the present study indicates that the facs - mn : zns qds can be synthesized entirely at room temperature and aqueous medium following simple wet chemistry method . a fast low cost process utilizes environmentally suitable materials such as cs and fa to stabilize the qds and to formulate the composite with targeted drug delivery and imaging functionalities . with that , this report not just provides information about the synthesis of targeted mn : zns qds , but also serves as a reference for the characterization of those qds by means of various instrumental techniques such as xrd , xrf , xps , uv - vis , ftir , hrtem , fesem , edax , tga , and fluorescence . from the results obtained so far , we are convinced that the doping of mn - atom impurities will significantly enhance the luminescence centers of zns nanocrystals and will widely support the considerable interest for the use and application of doped zns qds as excellent materials for targeted fluorescence imaging studies . of greater interest , the in vitro cytotoxicity study of the composite shows that the qds are nontoxic to human breast cell lines and demonstrated specific cellular uptake by fr - expressing cancer cells , emitting fluorescence efficiency in the process . thus , following the effective encapsulation of the qds with biodegradable polymer and conjugated with a targeting ligand , the as - synthesized facs - mn : zns composite can further support the ongoing effort toward the effective targeted drug delivery and imaging of tumor - bearing cells . benign prostatic hyperplasia ( bph ) is found in over half of 60-year - old men and in almost all 80-year - old men who develop bladder outlet obstruction ( boo ) and lower urinary tract symptoms ( luts ) . boo is the initial pathophysiological change caused by an enlarged adenoma and is followed by detrusor overactivity ( do ) or underactivity ( dua ) . the degree of boo is an important factor that can reflect the severity of disease and that can aid in choosing a treatment method as well as in measuring the outcome of the treatment . it has been shown that one third of male patients with luts do not have definite boo and that 5 to 35% of the patients with luts and undefined boo do not have favorable symptom recovery after transurethral resection of the prostate ( turp ) [ 2 - 7 ] . relevant examinations ranging from serum prostate - specific antigen ( psa ) to urodynamics can all reflect different aspects of the severity of bph . urodynamics is the only method , however , that can quantify the degree of boo and the status of detrusor contractility . therefore , guidelines from the international scientific committee and the american urological association on the management of bph both recommend the use of urodynamics to evaluate bph patients considered as candidates for invasive therapy . however , the routine use of preoperative urodynamics is still a controversial point in published articles because of the invasiveness and high costs of the method . in this research , we attempted to determine whether other parameters could be used to measure the severity of boo through less - invasive or noninvasive examinations by analyzing correlations among parameters from clinical history , symptoms , ultrasonography , and urodynamics . this retrospective analysis was conducted on patients with bph who had received either medication or surgical treatment at this hospital between may 2010 and june 2011 . the therapeutic decision for turp all patients were evaluated with the international prostate symptom score ( ipss , including the total score ; subtotal score of storage symptoms comprising the summation of nocturia , urgency , and an increased frequency score ; and subtotal score of voiding symptoms comprising the summation of hesitancy , intermittency , and weak stream score ) and quality of life ( qol ) questionnaires in addition to undergoing basic clinical evaluations ( medical history , physical examination , urinalysis , and renal function assessment ) before treatment . free flowmetry measurement was performed for all patients with the result being adopted when the voiding volume was more than 150 ml . urodynamics was performed only for patients needing surgery by use of a multichannel system ( uds64-iii , laborie co. , quebec , canada ) . first , water - filling cystometry was done with the patients in the supine position with the use of a transurethral 12 fr double - lumen catheter and the simultaneous monitoring of rectal pressure . filling was performed at a rate of 50 ml / min with normal saline and was stopped if the patient had a strong desire to void . pressure flow study ( pfs ) was then performed by asking the patients to void in an upright position with a suprapubically placed 6 fr cystostomy tube to monitor the bladder pressure . maximum urinary flow ( qmax ) and pressure of the detrusor at qmax ( pdetqmax ) were recorded . the bladder outlet obstruction index ( booi , by " pdetqmax-2qmax " ) and the bladder contractility index ( by " pdetqmax+5qmax " ) were then calculated by use of equations from the ics . total prostate volume ( tpv ) , transitional zone volume ( tzv ) , intravesical prostatic protrusion ( ipp ) , and post - voiding residual ( pvr ) were measured by transrectal ultrasonography ( trus ) with the transitional zone index ( tzi , by " tzv / tpv " ) being calculated for all patients . the exclusion criteria for enrolled subjects were 1 ) booi less than 20 or qmax more than 20 ml / s , 2 ) disease with boo other than bph , 3 ) history of prostatic or urethral surgery , 4 ) diagnosed carcinoma of the prostate or bladder , 5 ) known bladder stones or neurogenic bladder dysfunction , and 6 ) not having taken standard medication involving both alpha - adrenergic blockers and 5-alpha - reductase inhibitors for over 6 months . the relevant clinical data of the subjects were recorded and classified by degree of ipp ( < 10 mm , 10 to 20 mm , and > 20 mm ) and tzi value ( < 0.5 , 0.5 to 0.7 , and > 0.7 ) . all quantitative variables were tested for the type of distribution by use of the one - sample kolmogorov - smirnov test . univariate analyses including one - way analysis of variance and kruskal - wallis test were used for variables with normal or skewed distributions , respectively , to assess the differences between patients receiving drug therapy and those undergoing surgical therapy and between patients with different degrees of ipp and tzi values . the -test was used for categorical variables to identify whether there were different incidences of acute urinary retention ( aur ) influenced by different ipp or tzi grade . finally , bivariate correlation and multiple regression analysis were used to assess correlations between parameters from trus and pfs . all analyses were performed by using the routines of the ibm spss ver . 19.0 ( ibm co. , new york , ny , usa ) , and the research attained ethical approval from the ethics committee of xin hua hospital , and all subjects gave written informed consent . a total of 365 patients were enrolled in the research study , with 203 patients receiving medication and the remainder having undergone turp . the clinical data of all subjects were classified according to therapy and degree of ipp and tzi and are listed in tables 1 and 2 . univariate analyses showed significant differences in the total , storage , and voiding scores of the ipss ; qol score ; tpv ; tzv ; tzi ; ipp ; and qmax between the therapy groups ( p<0.05 ) . baseline total prostate - specific antigen , tpv , tzv , ipp , qmax , pdetqmax , and booi classified by different degrees of tzi were also found to be significantly different ( p<0.05 ) . differences were also found in the voiding symptom score , tpv , tzv , tzi , qmax , and booi classified by degree of ipp ( p<0.05 ) . ipp ( p=0.000 ) and tzi ( p=0.000 ) both had statistically significant effects on the cause of aur ( table 2 ) by -test . the bivariate correlation analysis of parameters from symptom score , trus , and urodynamics showed significant correlations between tzi and qmax ( r=-0.887 , p=0.001 ) , pdetqmax ( r=0.725 , p=0.028 ) , and booi ( r=0.508 , p=0.029 ) and between ipp and voiding symptom score ( r=0.353 , p=0.033 ) , qmax ( r=-0.852 , p=0.014 ) , and booi ( r=0.469 , p=0.042 ) . multiple regression analysis in the subjects who underwent turp showed that both tzi and ipp had significant correlations with qmax and booi , whereas tzi had a significant correlation with pdetqmax ( table 3 ) . prostatic adenoma enlargement increases urethral resistance and leads to boo , further resulting in compensatory changes in bladder function . however , the elevated detrusor pressure required to maintain urinary flow in the presence of increased outflow resistance occurs at the expense of normal bladder storage function , which is the source of do . with the continuation of obstruction , decompensation of the detrusor and dua the degree of boo is correlated with the severity of obstruction - relevant symptoms , and the recovery of boo is used to evaluate the efficacy of treatment of bph . furthermore , the baseline degree of boo was recently found to influence the outcome of treatment . research has shown that patients with boo have better outcomes from turp than do those without boo [ 11 - 14 ] . one research study showed that patients with boo will still have a favorable surgical outcome even if they have do or dua . therefore , some hospitals use urodynamics as a routine preoperative examination to confirm whether the candidates have explicit boo and good detrusor contractility . however , urodynamic study is not totally innocuous , with significant evidence of discomfort and urinary infections associated with performing the examination , as well as imposing additional cost to the patient or to the institution . for this reason , some research has been initiated to find less - invasive or noninvasive examinations for evaluating the degree of boo . ipp measured as the shortest distance connecting the protruded end of the prostate into the bladder based on the bladder neck in the sagittal plane reflects the maximum longitudinal length of the prostate and may help in assessing the obstructive level of the prostate . first studied the correlation between ipp and the booi in 30 male outpatients in 2005 and found that ipp grading correlated well with the booi . analyzed 206 bph patients classified by different ipp grade and found that the ipp value positively correlated with tpv , psa , pvr , qmax , pdetqmax , and booi as well as the incidence of aur , bladder trabeculation , detrusor overactivity , and low bladder compliance . analyzed 260 men with luts and found that the booi was higher in patients with apparent ipp than in those without . the tzi calculated as tzv divided by tpv may also correlate with the obstructive level because higher volumes of the transition zone will result in harder pressure on the urethra . kaplan et al . evaluated 61 men with symptomatic bph and found a significant correlation between tzi and symptoms , qmax , and pdetqmax . analyzed 116 bph patients and found that tzi and tzv were both positively correlated with booi and ipss . milonas et al . reported that lower tzi was an independent predictor of ineffective surgical outcome . in the present study , we found significant correlations in surgical patients between both tzi and ipp with parameters reflecting the level of boo , such as qmax , pdetqmax , and booi . these results are consistent with the results of former research and suggest that tzi and ipp may be appropriate parameters in diagnosing and classifying boo . for a long period , turp has been the gold standard surgical procedure based on the concept of removing the whole enlarged adenoma involved in static and dynamic urethral obstruction . however , the development of medication such as alpha - adrenergic blockers and 5-alpha - reductase inhibitors has decreased the progression of bph and the operation rate in patients in recent years . however , some patients can not achieve favorable recovery from drug therapy and need a surgeon to relieve the symptoms . the patients needing surgical therapy in our research were found to have higher values of both ipp and tzi than the patients needing only drug therapy . this result suggests that ipp and tzi could measure the disease progression in bph patients receiving medication and might have predictive value for medication efficacy . higher ipp grade was found to correlate with higher voiding symptom score , which demonstrated that ipp could reflect the severity of bph from not only an objective aspect but also a subjective aspect . aur is one of the most serious complications of bph and an indication for surgical intervention . this research has found positive correlations between not only ipp but also tzi and the incidence of aur , which further suggests that these 2 factors might be used to predict the progression of bph and the possibility of undergoing surgical therapy . in general , this research investigated tzi and ipp from trus in bph patients and found positive correlations between these indexes and symptoms , boo level , and the incidence of aur . tzi and ipp were also found to differ significantly between bph patients receiving medication and those undergoing surgical therapy . the results demonstrated that the two parameters had favorable value for assessing severity and progression in patients with bph . however , this research was retrospective only , with inevitable bias from subject selection and follow - up time . therefore , more prospective research should be launched to investigate the predictive value of the two parameters for the progression and treatment efficacy of bph .

in this study , we synthesized a multifunctional nanoparticulate system with specific targeting , imaging , and drug delivering functionalities by following a three - step protocol that operates at room temperature and solely in aqueous media . the synthesis involves the encapsulation of luminescent mn : zns quantum dots ( qds ) with chitosan not only as a stabilizer in biological environment , but also to further provide active binding sites for the conjugation of other biomolecules . folic acid was incorporated as targeting agent for the specific targeting of the nanocarrier toward the cells overexpressing folate receptors . thus , the formed composite emits orange red fluorescence around 600 nm and investigated to the highest intensity at mn2 + doping concentration of 15 at.% and relatively more stable at low acidic and low alkaline ph levels . the structural characteristics and optical properties were thoroughly analyzed by using fourier transform infrared , x - ray diffraction , dynamic light scattering , ultraviolet - visible , and fluorescence spectroscopy . further characterization was conducted using thermogravimetric analysis , high - resolution transmission electron microscopy , field emission scanning electron microscopy , energy dispersive x - ray spectroscopy , x - ray fluorescence , and x - ray photoelectron spectroscopy . the cell viability and proliferation studies by means of mtt assay have demonstrated that the as - synthesized composites do not exhibit any toxicity toward the human breast cell line mcf-10 ( noncancer ) and the breast cancer cell lines ( mcf-7 and mda - mb-231 ) up to a 500 g / ml concentration . the cellular uptake of the nanocomposites was assayed by confocal laser scanning microscope by taking advantage of the conjugated mn : zns qds as fluorescence makers . the result showed that the functionalization of the chitosan - encapsulated qds with folic acid enhanced the internalization and binding affinity of the nanocarrier toward folate receptor - overexpressed cells . therefore , we hypothesized that due to the nontoxic nature of the composite , the as - synthesized nanoparticulate system can be used as a promising candidate for theranostic applications , especially for a simultaneous targeted drug delivery and cellular imaging . purposethe aim of this research was to assess the value of the transitional zone index ( tzi ) and intravesical prostatic protrusion ( ipp ) from transrectal ultrasonography in evaluating the severity and progression of disease by analyzing the relationship between the 2 parameters and symptoms , clinical history , and urodynamics in benign prostatic hyperplasia ( bph ) patients undergoing different treatment.materials and methodsa total of 203 patients receiving medication and 162 patients who underwent transurethral resection of the prostate because of bph were enrolled in this retrospective analysis . the clinical history and subjective and objective examination results of all patients were recorded and compared after being classified by tzi and ipp level . linear regression was used to find correlations between ipp , tzi , and urodynamics.resultsthe 2 parameters were found to differ significantly between patients receiving medication and patients undergoing surgical therapy ( p<0.05 ) . psa , maximum flow rate ( qmax ) , detrusor pressure at qmax ( pdetqmax ) , and the bladder outlet obstruction index ( booi ) differed according to various tzi levels ( p<0.05 ) . in addition , the voiding symptom score , qmax , and booi of subgroups with various ipp levels were also significantly different ( p<0.05 ) . both tzi and ipp had significant effects on qmax , booi , and pdetqmax ( p<0.05 ) and the incidence of acute urinary retention ( p=0.000).conclusionsthe results demonstrated that both tzi and ipp had favorable value for assessing severity and progression in patients with bph . further studies are needed to confirm whether the two parameters have predictive value in the efficacy of bph treatment and could be considered as factors in the selection of therapy .

apert syndrome ( acrocephalosyndactylia ) is a developmental malformation characterized by craniosynostosis , a cone - shaped calvarium ( acrocephaly ) , hypertelorism , midface hypoplasia , pseudo cleft - palate , a parrot beak - shaped nose , pharyngeal attenuation , and syndactyly of the hands and feet.1 - 3 the inheritance of apert s syndrome is autosomal dominant with the locus of a mutation of fgfr2 on chromosome 10q ( 10q2526 ) . suture progenitor cells with fibroblast growth factor receptors ( fgfr2 ) that have undergone a mutation can not transduce signals from extracellular fibroblast growth factors ( fgfs ) . therefore , these cells do not receive the signal to produce the necessary fibrous material essential for a normal calvarial suture.3 apert syndrome was first reported by wheaton in 1894 and french pediatrician eugene apert published a series of nine cases in 1906.1,4 most cases are sporadic , with an incidence of 1:160 000 ; however due to high infant mortality , the incidence in the general population is lower . advanced male parental age has been consistently noted.5 during the course of the disease , growth and mental retardation can be observed.1,5 in apert cases , the spheno - occipital and spheno - ethmoidal synchondroses and the fronto - ethmoidal suture fuse early , resulting in a severely shortened posterior cranial base and a relatively short anterior cranial base with a resultant hypoplastic midface . consistent with the observation of midface hypoplasia , the maxilla also exhibits a transverse deficiency.3 the most readily observed malocclusions are a severe maxillary anterior open bite and a severely crowded and retrusive maxillary arch due to the constricted secondary palate.3 the maxillary alveolar arch is v - shaped.6 due to the narrower maxillary arch , bilateral or unilateral posterior crossbite can be observed.4,7 impactions , severe crowding of developing teeth within the alveolus , delayed eruption , thick gingiva , and sometimes supernumerary teeth or congenitally missing teeth are the hall marks of maxillary dental development in apert patients.8 the nasopharyngeal and oropharyngeal attenuation cause apert s individuals to become mouth breathers with a resultant anterior open bite.3 a 16-day - old female infant was admitted to the department of orthodontics of selcuk university because of cleft palate . from her parents history she was the second child from a normal mother s third pregnancy of a consanguineous marriage between cousins . apert syndrome was diagnosed by dna analysis and physical examination in the medical faculty . during extraoral examination of the infant , whose appearance was noted with defects , it was observed that he displayed a cone - shaped calvarium , midface hypoplasia , hypertelorism , ocular proptosis , shallow orbits , down - slanting lateral canthi and palpebral fissures , a depressed nasal bridge , low - set ears , and syndactyly of the fingers and toes ( excluding the thumbs ) ( figures 1 , 2 , and 3 ) . intraoral clinical examination revealed that upper and lower alveolar bases were normal , and a bifid uvula and secondary cleft palate ( figures 4 , and 5 ) were presented . the maxillary impression was taken by using silicon - based impression material ( zhermack spa , badia polesine , italy ) and an orthodontic plaster model was obtained ( figures 4 , and 5 ) . after the cleft was covered with the wax , the acrylic appliance was made ( figure 6 ) . feeding the baby and orientating the growth was the goal of using this appliance for treatment.9 the appliance was worn on the patient , and the parents were instructed in full - time wear and cleaning procedures of the appliance . the parents was also informed about feeding the baby in a vertical position , using a nipple with a small hole through which milk flow rate was in the form of intermittent drops ; therefore , the infants s perioral and buccal muscles would strengthen . the baby was checked at intervals of six weeks for a period of six months ( figure 5 ) . when the baby was eight months , an operation was done to correct anomalies in the skull . a silicon - based impression material was preferred to make the maxillary impression easier . during the impression the baby s head is tilted backwards by holding the left knee forward.9 it is postulated that mutation in the frfr2 gene has an effect on the mesenchymal development , which has an effect on tooth morphogenesis.10 many oral manifestations can be attributed to the presence of this mutation . failure in the anteroposterior and downward growth of the maxilla causes the maxillary hypoplasia and a resultant contraction of nasopharyngeal airway.11 therefore , one should pay attention to obstructive sleep apne syndrome and premature death.1 in patients with apert syndrome , severe skeletal class iii open bite malocclusion can be observed due to the maxillary deficiency and the inclination of the upper jaw . the infant is likely to suffer from oral hygiene problems during treatment . for the patient with apert syndrome , the new generation of electric tooth brushes and fluoride mouth rinses may make the task easier . professional care -including frequent dental examinations , oral hygiene prophylaxis , fluoride treatments , and dental sealants- are very important.4 tosun and sener s study showed that apert syndrome was in parallel with g6pd deficiency.4 g6pd deficiency is an enzymatic hereditary disorder leading to hemolytic anemia as a result of red blood cell destruction . the main problem in g6pd deficiency is that hemolysis can be precipitated by a number of factors , such as oxidant drugs , eating fava beans , or intercurrent infection . drugs that may induce hemolysis include sulphonamides , chloramphenicol , aspirin , acetaminophen , penicillin , and streptomycin . therefore , the dentist must avoid drugs that may potentially induce hemolysis as result of g6pd deficiency.4 a significant proportion of patients with apert syndrome has mental retardation . in these patients , significant social problems , speech difficulties , and attention deficit are noted.1 in apert syndrome , definite diagnosis can be made by dna analysis . crouzon syndrome -another craniosynostosis disorder- is the result of the same gene mutation occurring in different locations.1 orthodontists can achieve an improvement in the patient s appearance and function of dentoskeletal structures by a combined orthodontic and orthognathic surgical treatment plan . electrostatic potentials and free energies were calculated with a modified version of the delphi program , adapted to solve the nonlinear poisson - boltzmann equation for protein - membrane systems . delphi yields finite - difference solutions to the poisson - boltzmann equation ( the fdpb method ) for a system where the solvent is described in terms of a bulk dielectric constant and concentrations of mobile ions , whereas the solutes ( here , the proteins and membrane ) are described in atomic detail by the coordinates of the individual atoms , their atomic radii , and their partial charges . the fdpb methodology has been applied to numerous peptide - membrane and protein - membrane systems , producing results that predict successfully and with high accuracy the following : 1 ) macroscopic properties , such as quantitative differences between the membrane interactions of homologous proteins and effects of lipid composition or ionic strength ; and 2 ) microscopic properties , such as the effect of point mutations or binding of metal ions on membrane interactions ( for examples see refs . 30 , 33 , 35 - 40 ) . atomic structural models of proteins and membranes the atomic model we used for the gt heterotrimer was based on the gt / i111 crystal structure ( pdb code 1got ) solved by lambright et al . because the g subunit in this structure is a chimera of gt and gi1 ( residues 216 - 294 of bovine gt were replaced with residues 220 - 298 of rat gi1 to improve protein expression ) , a composite model was built for heterotrimeric gt by manually copying the backbone and side chains of these residues from the corresponding region of native gt , taken from the gt-gdp structure ( 1tag ) ( 41 ) . the structure of these two corresponding regions is highly similar ( 16 ) , and their backbone root mean square deviation is 0.5 . the structure of bovine g11 ( pdb code 2trc ) ( 42 ) was used in calculations for dissociated g11 , as in a previous study ( 30 ) . both g1 termini in the heterotrimeric model , which are slightly shorter than in the structure of dissociated g11 , were extended by manually copying the extra residues from the structure of dissociated g11 . the n terminus of gt , in its heterotrimeric form , is in an extended helical conformation and is stabilized by interactions with g11 ( see supplemental fig . after dissociation from g11 , this subdomain does not have any significant stabilizing interactions with the membrane ( supplemental fig . available structures of monomeric gt are not applicable to our calculations because they lack the entire n terminus , which was cleaved prior to crystallization . however , we note that the n terminus in the crystal structure of the homologous gi1-gdp ( 68% sequence identity to gt ) refolds onto the bulk of the protein to form a compact subdomain that is stabilized by a network of interacting residues that are conserved between gi1 and its homologs go and gt ( 43 ) . a folded and highly ordered gi1 n terminus was also shown for the myristoylated form of gi1 in solution , and it was suggested that this feature is conserved in homologous g subunits ( 44 ) . we therefore assumed that soluble gdp - bound gt adopts a similar conformation , and we modeled it on the structure of monomeric gi1-gdp ( pdb code 1gdd ) using the program nest ( 45 ) and remodeled unconserved side chains using scap ( 46 ) . hydrogen atoms were added to the protein structures with the program charmm , and the structures were subjected to conjugate gradient minimization with a harmonic restraint force of 50 kcal / mol / applied to the heavy atoms located at the original crystallographic coordinates . hydrogens were added to the guanine nucleotides bound to the g subunits using the builder module in insight ii ( accelrys ) . phospholipid bilayers of lateral dimensions 165 172 and ratios of 0:1 , 1:8 , 1:5 , 1:3 , 1:2 , and 1:1 acidic lipid ( phosphatidylserine ) to neutral lipids ( phosphatidylcholine ) were built as described in previous work ( 30 , 33 - 37 , 39 , 47 ) . in vivo , the distribution of acidic lipids is known to be asymmetric across the bilayer , and following previous studies ( 30 , 33 , 35 - 40 , 48 , 49 ) , we assume that all of the phosphatidylserine is located on the cytosolic leaflet of the rod outer segment membranes . except where specified otherwise , in our calculations we used bilayers with a ratio of 1:2 phosphatidylserine / phosphatidylcholine ; this ratio has been assumed to be a reasonable approximation for the lipid composition facing the transducin under physiological conditions ( 30 ) , and indeed , as shown in fig . 4 and discussed below , this membrane composition is representative of the wider range of possible compositions . as in previous work , we assume that the bilayer does not change structure or position when it interacts with the protein ( see ref . the protein / membrane model was mapped onto a fine three - dimensional grid , where each small cube represents a small region of the peptide , membrane , or solvent . as in previous studies of comparable protein - membrane systems ( 30 , 33 - 37 , 39 ) , we took the charges and radii used for the amino acids from the charmm22 parameter set and for the lipids from ref . regions inside the molecular surfaces of the protein and membrane were assigned a dielectric constant of 2 , and those outside were assigned a dielectric constant of 80 , combined with an ion exclusion layer of 2 around the solute . unless stated otherwise , the salt concentration was set to 100 mm , as in previous studies ( 30 , 33 , 35 - 40 ) . the numerical calculation of the potential was iterated to convergence , defined as the point at which the potential changes less than 10 kt / e between successive iterations . a sequence of focusing runs of increasing resolution was employed to calculate the electrostatic potentials ( e.g. 0.3 , 0.6 , 1.2 , and 2.4 grid / ) . electrostatic energies were obtained using the calculated potentials , and the electrostatic energy of a protein - membrane interaction was determined as the difference between the following : 1 ) the electrostatic free energy of the protein in a specific orientation and distance relative to the membrane surface ; and 2 ) the electrostatic free energies of the protein and membrane infinitely far apart ( i.e. calculated separately ) . the numerical error of the free energy calculations , estimated by the difference between the calculations of the two highest resolution scales , was < 0.1 kcal / mol in all calculations . the nonspecific electrostatic interactions calculated here are relatively insensitive to local changes in conformation . different combinations of composite models from two separate pdb structures or different minimization protocols , which produced global root mean square deviations of up to 2 between models , changed the calculated electrostatic energies of interactions by less than the numerical error for a given protein - membrane complex . global sampling of transducin orientations to calculate transducin - membrane interactions previous studies used visual inspection to find an approximate minimum free energy orientation of a protein relative to the membrane and then sampled extensively close to this orientation to find the minimum free energy orientation ( 30 , 33 - 37 ) . here , however , visual inspection could not reliably determine the approximate minimum - energy orientation of the gt heterotrimer and gt with respect to the membrane , because these proteins do not have prominent positively charged patches that clearly determine the orientation of minimum energy . we therefore implemented a global sampling of all nonredundant euler rotations , rotating the protein around the point in the structure closest to the lipid attachment point . this point corresponds to the n terminus in gt calculations , the c terminus of g1 in g11 calculations , and the midpoint between them for the heterotrimer structure ( where the two termini are 15 apart ) . for each orientation , the membrane was moved so as to place the molecular surfaces of the protein and membrane 3 apart ( r = 3 ) , and the electrostatic energy was calculated as above . r = 3 was chosen because at orientations where the electrostatic interaction is attractive , the energy value at this distance is minimal ( see supplemental material and refs . 30 , 33 , 35 , 40 ) . combining lipid anchor constraints with global orientation sampling the transducin structures used here do not have coordinates for the lipid anchors nor for the termini to which the anchors are covalently attached as follows : the myristoyl - gaga- at the n terminus of gt and the -ggc - farnesyl at the c terminus of g1 . for each orientation we assumed that a lipid anchor can reach the membrane if the measured distance between each relevant terminus and the membrane was equal to or less than 13 for gt and 10 for g1 , thresholds corresponding to the length of the fully extended linkers . 16 , based on their amino acid sequence ) and able to adopt fully extended conformations . when we determined that a lipid anchor can reach the membrane in a specific orientation , we assigned to this orientation an energetic contribution of -6 kcal / mol , which corresponds to the estimated energetic contribution of an individual lipid modification ( see under discussion for more details on these estimates ) . when both lipid anchors could reach the membrane , we assigned to such an orientation an energetic contribution of -12 kcal / mol . calculation of average energy and entropy previous work established that when the peripheral association between a protein and a membrane is of substantial attractive nature , the relative binding energies and the electrostatic contribution to binding are well approximated by the energy value at the minimum free energy orientation ( 30 , 33 - 37 , 39 ) . in addition to finding the minimal free energy value , we also calculated the boltzmann - weighted average energy , as in ref . 37 , and see equation 1 , where k is the boltzmann constant , and t is the absolute temperature . the calculations were repeated twice , with and without the constraints of the lipid anchor . the latter can show whether specific orientations provide sufficient attraction to the membrane to compensate for the energetic penalty associated with removing the lipid anchor out of the bilayer ( see above ) , and represent the effect of electrostatics on the soluble forms of the proteins ( i.e. before the lipid anchors attach to the membrane ) . we used the bootstrapping re - sampling method to check the statistical significance of our results ( as in ref . . the free energy cost because of the entropic penalty associated with limiting gt rotations was approximated by equation 2 , where s2 is the entropy of gt with the additional constraints ( lipid anchor and/or electrostatics ) , and s1 is the reference state ( having the same number of microstates but with an equal probability of being in each state and with all of these probabilities summing to unity ) . we estimated the error in the entropy calculation by adding a random value ( in the range of the numerical error , 0.1 kcal / mol ) to the calculated free energies and repeated the entropy calculations 100 times , reaching an estimated error of 0.02 kcal / mol . figure 1.interactions of the gt heterotrimer with the membrane . a , global orientation sampling of the gt heterotrimer . the x ( ) and y axes ( ) mark euler angles of rotation from the initial orientation ( supplemental fig . the z axis and the color code both mark the calculated electrostatic free energy of interaction with the membrane ; positive ( red ) energies will result in repulsion , whereas negative ( blue ) energies will result in attraction . the magenta and cyan lines enclose lipid - allowed orientations , which enable the g1 ( magenta ) and the gt ( cyan ) lipid anchors to reach the membrane . representative gt orientations are marked on the plot of global orientation sampling for gt , with letters corresponding to subsequent panel captions . b , gt orientation of minimum free energy , which is representative of tilted orientations ; gel = -0.8 kcal / mol . c , electrostatic potential map of the gt heterotrimer in the orientation of minimum free energy ( the same orientation as in b ) . electrostatic potential maps were visualized by drawing equi - potential contour meshes for the values + 1 kt / e ( 25mv , blue mesh ) and -1 kt / e ( -25 mv , red mesh ) . gt subunits are shown in connolly surface representation and colored pink ( ) , light green ( ) , and light blue ( ) , and the functionally relevant termini are annotated . two large negatively charged patches on gt and g11 are marked with orange and red arrows , respectively ; these patches cause the repulsion observed in the lipid - allowed orientations at 0 and > 60. d , representative parallel orientation ; gel = 1.1 kcal / mol . e , lipid - allowed orientation with moderate repulsive electrostatics ; gel = 1.8 kcal / mol . f , lipid - disallowed orientation , where the g1 lipid anchor can not reach the membrane ; gel = 4 kcal / mol . g , lipid - disallowed orientation , where both lipid anchors can not reach the membrane ; gel = 3.5 kcal / mol . b and d - g , gt subunits are shown in worm representation and are colored red ( ) , green ( ) , and blue ( ) . the n - terminal helix of gt is colored magenta , and the c terminus of gt is colored orange . interactions of the gt heterotrimer with the membrane . a , global orientation sampling of the gt heterotrimer . the x ( ) and y axes ( ) mark euler angles of rotation from the initial orientation ( supplemental fig . the z axis and the color code both mark the calculated electrostatic free energy of interaction with the membrane ; positive ( red ) energies will result in repulsion , whereas negative ( blue ) energies will result in attraction . the magenta and cyan lines enclose lipid - allowed orientations , which enable the g1 ( magenta ) and the gt ( cyan ) lipid anchors to reach the membrane . representative gt orientations are marked on the plot of global orientation sampling for gt , with letters corresponding to subsequent panel captions . b , gt orientation of minimum free energy , which is representative of tilted orientations ; gel = -0.8 kcal / mol . c , electrostatic potential map of the gt heterotrimer in the orientation of minimum free energy ( the same orientation as in b ) . electrostatic potential maps were visualized by drawing equi - potential contour meshes for the values + 1 kt / e ( 25mv , blue mesh ) and -1 kt / e ( -25 mv , red mesh ) . gt subunits are shown in connolly surface representation and colored pink ( ) , light green ( ) , and light blue ( ) , and the functionally relevant termini are annotated . two large negatively charged patches on gt and g11 are marked with orange and red arrows , respectively ; these patches cause the repulsion observed in the lipid - allowed orientations at 0 and > 60. d , representative parallel orientation ; gel = 1.1 kcal / mol . e , lipid - allowed orientation with moderate repulsive electrostatics ; gel = 1.8 kcal / mol . f , lipid - disallowed orientation , where the g1 lipid anchor can not reach the membrane ; gel = 4 kcal / mol . g , lipid - disallowed orientation , where both lipid anchors can not reach the membrane ; gel = 3.5 kcal / mol . b and d - g , gt subunits are shown in worm representation and are colored red ( ) , green ( ) , and blue ( ) . the n - terminal helix of gt is colored magenta , and the c terminus of gt is colored orange . we translated differences in the free energy of interaction to changes in association / dissociation rates ( k ) using equation 3 , ( eq.3)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}\;\;{\delta}k\hspace{1em}=\hspace{1em}e^{-{\delta}gi / k_{b}t}\end{equation*}\end{document } visualizing three - dimensional potential maps three - dimensional potential maps were visualized by mapping the values onto the molecular surface of the protein and by drawing equi - potential contour meshes that connect all the points in space having a specific potential value ( here 1 kt / e , which are equal to 25 mv ) . characterizing the interaction of the gt heterotrimer with the lipid bilayer we analyzed the interaction of gt with the membrane using global orientation sampling , using the fdpb method to calculate the electrostatic free energy of interaction at each orientation ( fig . at each orientation , we also measured whether each of the two lipid anchors can reach the lipid bilayer ( marked as lipid - allowed orientations in fig . these measurements show that 11% of all possible gt orientations enable both lipid anchors to reach the membrane . by calculating the effects of electrostatics and the lipid anchors separately , we can examine their individual contributions and analyze cases in which only one of these factors is significant for membrane interactions ( e.g. when soluble transducin targets back to the membrane from the cytosol , or when transducin interacts in vitro with neutral membranes ) . when we determined that a lipid anchor can reach the membrane in a specific orientation , we assigned to this orientation an energetic contribution of -6or -12 kcal / mol , corresponding to the estimated energetic contribution of one or two lipid modifications , respectively ( see discussion for more details on these estimates ) . we assumed that a lipid anchor can reach the membrane when the distance between the bilayer and the relevant terminus in the structure was equal to or less than predetermined distance thresholds ( see experimental procedures ) . this assumption considers the terminal 3 - 4 amino acids that connect to the lipid anchors and are not present in the crystal structure as conformationally flexible linkers that are able to fully extend , as was suggested previously based on their amino acid sequence ( 16 ) . nevertheless , our results are not dependent on the exact values of these distance thresholds ; because of the large size of gt , its geometry , and the close proximity of the two relevant termini , increasing or decreasing these distance thresholds by up to 50% resulted in essentially the same ensemble of lipid - allowed orientations . only a minor ensemble of orientations ( 6% ) exhibits either a small attraction ( 3% of all orientations ) or a weak repulsion . we label orientations with an interaction energy ( gel ) within 2 kcal / mol of the minimum electrostatic free energy ( -0.8 kcal / mol , see table 1 ) , as electrostatically favorable orientations . we chose this threshold because it corresponds to a substantial ( 30-fold ) decrease in the occupation probability with respect to the energy minimum . ( note that the results are relatively insensitive to the precise value of the threshold because the selected value is far enough from the minimum so that changing it by 0.5 kcal / mol has a small effect on which orientations are labeled as favorable . ) the electrostatically favorable orientations of gt are clustered around the orientation of minimum electrostatic free energy ( fig . a small positively charged patch around the attachment points of the two lipid anchors is proximal to the membrane , whereas the negative potential around most of gt is positioned further away from the membrane ( e.g. fig . this results in a weak attraction at the orientation of minimum electrostatic free energy ( -0.8 kcal / mol ) and an even smaller average energy of interaction ( -0.3 kcal / mol , see table 1 ) . table 1minimum and average electrostatic free energies of interaction of heterotrimeric / dissociated transducin with the lipid bilayerthe electrostatic free energy values are in kcal / mol , and the numerical error is < 0.1 kcal / mol.transducin stategel , min ( lipid anchor 's in membrane)gel , min ( all possible orientations)a<gel > b(lipid anchor / s in membrane)<gel > b(all possible orientations)a 0.8 0.8 0.3 ~0 3.2 3.2 2.8 2.7 + 1.8 1.2 + 2.2 0.4 acalculated by sampling of all possible orientations ( i.e. without the constraints of the lipid anchor / s).bboltzmann - weighted averaged energies ( see experimental procedures for details ) . minimum and average electrostatic free energies of interaction of heterotrimeric / dissociated transducin with the lipid bilayer the electrostatic free energy values are in kcal / mol , and the numerical error is < 0.1 kcal / mol . calculated by sampling of all possible orientations ( i.e. without the constraints of the lipid anchor / s ) . boltzmann - weighted averaged energies ( see experimental procedures for details ) . fig . the orientation of minimum free energy ( fig . 1 , b and c ) places the n - terminal helix of gt at an angle of 30 to the membrane surface and is similar to the tilted orientations suggested previously ( see above ) . 1d ) position negatively charged patches near the c terminus of gt ( marked with an orange arrow in fig . 1c ) close to the membrane and therefore result in electrostatic repulsion . because of this repulsion , parallel orientations are less favorable than the orientation of minimum free energy ( a tilted orientation ) by 2 kcal / mol , which corresponds to 30-fold difference in occupancy probability . furthermore , large negatively charged patches on g11 ( marked with a red arrow in fig . 1c ) oppose rotations of gt to orientations that position the n - terminal -helix of gt at angles > 60 to the membrane surface ( fig . 1 , a and e ) . these electrostatic repulsions limit gt rotation and constrain the gt c terminus , which is essential for binding activated rhodopsin , to face the membrane surface in all of the allowed orientations of gt . to estimate at the ensemble level how rotational electrostatic and/or lipid anchor constraints affect membrane - associated gt , we calculated the difference in the entropic component of the rotational free energy that results from these constraints . the reference state was a membrane - attached gt that can occupy all possible orientations with an equal probability ( i.e. without any rotational constraints ) . note that the effect on the degrees of freedom of gt does not depend on the strength of the gt - membrane interactions in specific orientations , but rather is a result of the global shape and characteristics of the energy - orientation landscape on gt ( as shown in fig . when we applied the rotational constraints of both lipid anchors , the entropic cost was 1.1 kcal / mol . applying only the constraint of the electrostatic interactions with the membrane results in an entropic cost of 1.3 kcal / mol ( with an estimated error of 0.02 kcal / mol , when these constraints were applied together , the entropic energy cost was 1.8 kcal / mol , i.e. electrostatics further enhance the effect of the lipid anchors by a factor of 3.5 . membrane interactions of dissociated g11electrostatic interactions of the dissociated gt and g11 with the lipid bilayer are very different from one another and from those of the transducin heterotrimer ( figs . 2 and 3 versus fig . 2a ) , we found a similar orientation of minimum electrostatic free energy ( with a similar gel = -3.2 kcal / mol ; table 1 ) as reported in a previous study ( 30 ) , which sampled around an initial orientation chosen by visual inspection . we also observed that , unlike the gt heterotrimer , g11 is attracted to the membrane in many orientations ( 18% of all possible orientations ) , allowing membrane - attached g11 to sample a wide range of dissimilar orientations that are both lipid - allowed and electrostatically favorable ( fig . 2 ) . furthermore , most of the electrostatically favorable orientations of g11 and particularly all orientations with gel less than -2 kcal / mol enable the lipid anchor to reach the membrane ( fig . the larger range of lipid - allowed orientations with significant electrostatic attraction to the membrane is also reflected in a stronger boltzmann - weighted average interactions energy ( -2.8 kcal / mol ; see table 1 for a detailed comparison ) . increasing or decreasing the distance threshold for determining whether the lipid anchor can reach the membrane by 50% had no effect on these results . additionally , when we removed the constraints of the lipid anchor and sampled all possible orientations of g11 , the electrostatic free energies of interaction did not change ( table 1 ) . taken together , these results suggest that the contributions of the lipid anchor and electrostatics to the free energy of g11 interaction with the membrane are independent of one another and therefore additive . figure 2.interactions of g11 with the membrane . a , global orientation sampling of g11 . the magenta line encloses lipid - allowed orientations , which enable the g1 c - terminal farnesyl to reach the membrane . representative lipid - allowed and electrostatically favorable g11 orientations are marked with letters corresponding to subsequent panel captions . b , g11 orientation of minimum free energy ; gel = -3.2 kcal / mol . c , electrostatic potential map of g11 in the orientation of minimum free energy ( the same orientation as in b ) , but rotated 90 around the z axis to better visualize the segregation of charges . g11 subunits and the lipid bilayer are depicted as in fig . 1 . b and d - f , the c terminus of g1 is marked with a black arrow , and the c terminus of g1 is marked with a magenta arrow . interactions of g11 with the membrane . a , global orientation sampling of g11 . the magenta line encloses lipid - allowed orientations , which enable the g1 c - terminal farnesyl to reach the membrane . representative lipid - allowed and electrostatically favorable g11 orientations are marked with letters corresponding to subsequent panel captions . b , g11 orientation of minimum free energy ; gel = -3.2 kcal / mol . c , electrostatic potential map of g11 in the orientation of minimum free energy ( the same orientation as in b ) , but rotated 90 around the z axis to better visualize the segregation of charges . g11 subunits and the lipid bilayer are depicted as in fig . 1 . b and d - f , the c terminus of g1 is marked with a black arrow , and the c terminus of g1 is marked with a magenta arrow . figure 3.interactions of gt with the membrane . a , global orientation sampling of gt. the cyan line encloses lipid - allowed orientations , which enable the gt n - terminal lipid anchor to reach the membrane . the lipid - allowed orientation with minimal free energy of interaction ( gel = 1.8 kcal / mol ) is marked with a black arrow . the lipid - disallowed orientation with minimal global electrostatic free energy ( -1.2 kcal / mol ) is marked with a yellow arrow . b , electrostatic potential map of gt in the lipid - allowed orientation with the minimal free energy of interaction , depicted as in fig . 1 . interactions of gt with the membrane . a , global orientation sampling of gt. the cyan line encloses lipid - allowed orientations , which enable the gt n - terminal lipid anchor to reach the membrane . the lipid - allowed orientation with minimal free energy of interaction ( gel = 1.8 kcal / mol ) is marked with a black arrow . the lipid - disallowed orientation with minimal global electrostatic free energy ( -1.2 kcal / mol ) is marked with a yellow arrow . b , electrostatic potential map of gt in the lipid - allowed orientation with the minimal free energy of interaction , depicted as in fig . membrane interactions of dissociated gtwithin the gt heterotrimer , the lipidated n terminus of gt assumes an extended helical conformation stabilized by interactions with g11 ( e.g. fig . however , this n - terminal helix is likely to refold into a compact conformation upon the dissociation of gt from g11 . although no available crystal structure of monomeric gt contains this part of the molecule , because of its proteolytic removal prior to crystallization , the crystal structure of the homologous gi1-gdp ( 43 ) does contain the n terminus , which is folded onto the bulk of gt and is stabilized by a network of interacting residues conserved between gi1 and gt ( and also go ) ( 43 ) . furthermore , a folded and highly ordered gi1 n terminus was shown for the myristoylated form of gi1-gdp in solution , and it was suggested that this structural feature is conserved in homologous g subunits ( 44 ) . we therefore proceeded with calculations for dissociated gt-gdp modeled on the structure of gi1-gdp ( 43 ) as a template ( fig . 3 ) . global orientation sampling of this gt conformation showed repulsion by the negatively charged membrane in most orientations ( fig . 3a ) , because the entire surface of gt is dominated by negative charges ( fig . 3b ) . in particular , all the lipid - allowed orientations of gt ( 11% of all possible orientations ) were repulsive . a few orientations that exhibited a small attraction to the membrane ( up to -1.2 kcal / mol ; see fig . 3a and table 1 ) placed the lipid attachment point on the distal face of gt relative to the membrane , and because of the energetic cost of removing the lipid anchor from the bilayer ( approximately -6 kcal / mol , see discussion ) , these orientations are disallowed . calculation of the electrostatic free energy of interaction of different gt states with bilayers of different acidic lipid composition . the vertical dashed line marks the lipid composition used in most calculations here ( 33% acidic lipids ) , whereas the two vertical dotted lines mark the range of possible lipid compositions facing transducin in rod outer segment disks ( see text for details ) . calculation of the electrostatic free energy of interaction of different gt states with bilayers of different acidic lipid composition . the vertical dashed line marks the lipid composition used in most calculations here ( 33% acidic lipids ) , whereas the two vertical dotted lines mark the range of possible lipid compositions facing transducin in rod outer segment disks ( see text for details ) . we quantified how the repulsive interactions , which characterize the lipid - allowed orientations of gt , affect its membrane affinity ( table 1 ) . for significant membrane attraction , the minimal free energy of interaction provides a good estimate and an upper bound to the membrane affinity of the protein ( 30 , 33 , 35 , 39 ) . however , for repulsive interactions , as in the case of monomeric gt , the magnitude of the minimal electrostatic repulsion in the lipid - allowed ensemble ( 1.8 kcal / mol ) provides a lower bound to the overall repulsion . because all the other lipid - allowed orientations are more repulsive , the boltzmann - weighted average of all gt lipid - allowed orientations is higher ( 2.2 kcal / mol ) , reducing the membrane affinity of gt in this conformation by 40-fold . a 50% increase in the distance threshold for determining whether the lipid anchor can reach the membrane had a small effect on these results ( < 0.2 kcal / mol ) . conversely , decreasing the distance threshold ( i.e. assuming the linker is not fully flexible ) resulted in significantly increased repulsion . dependence of the electrostatic interactions on distance and membrane composition we investigated whether the choice of protein - bilayer distance ( 3 ) influenced our major conclusions . calculation of the free energy of interaction as a function of distance showed a weak dependence ( supplemental fig . s3 ) , as long as the surfaces of the gt subunits and the membrane are 3 apart , a likely assumption given their highly charged nature and the strong repulsion at distances <3 ( see refs . 30 , 33 , 35 - 40 ) . as in previous work ( 30 ) , we used a bilayer model containing 33% acidic lipids to reproduce the bulk electrostatic properties of the cytosolic leaflet of disk membranes that faces transducin . previous investigations reported various proportions of acidic to neutral lipids in rod outer segment membranes ( 51 - 54 ) with the percentage of acidic lipids ranging from 13 to 20% . the distribution of acidic lipids is known to be asymmetric across the bilayer , and previous studies assumed that all of the phosphatidylserine is located on the cytosolic leaflet of the membrane ( 30 , 33 , 35 - 40 , 48 , 49 ) . accordingly , the percentage of acidic lipids facing gt can range from 25 to 40% . 4 shows that within this range , the membrane composition has almost no effect on the electrostatic interaction of the charged bilayer with gt / g11/gt in their minimal free - energy orientation ; these different membrane compositions change the calculated electrostatic interaction by less than 0.1 kcal / mol for gt and less than 0.3 kcal / mol for gt and g11 . dependence of the electrostatic interactions on ionic strength and implications for transducin purification protocols the membrane interactions of the transducin heterotrimer and its dissociated subunits depend on the ionic strength , with each of the three molecules exhibiting a unique salt dependence ( fig . 5 ) . increasing the salt concentration from 100 mm , at which our calculations were performed , to the physiological conditions of 150 mm , changes the membrane interaction energies of transducin and its subunits by < 10% ( < 0.03 kcal / mol for the heterotrimer , < 0.2 kcal / mol for gt , and < 0.3 kcal / mol for g11 ) and therefore does not significantly affect our major conclusions . in contrast , hypotonic conditions change the interaction energies for all three species dramatically . the small attraction of the gt heterotrimer to the membrane at physiological ionic strength changes to a strong repulsion under hypotonic conditions . as shown previously ( 30 ) , the electrostatic interaction of dissociated g11 exhibits a parabolic dependence on the ionic strength , with a maximal attraction at 50 mm salt and a reduction in affinity under more extreme hypotonic conditions . unlike g11 , gt is electrostatically repelled by the membrane at physiological ionic strength , and this repulsion increases considerably when the ionic strength is reduced ; a 10-fold reduction increases the repulsion to 8 kcal / mol , more than the membrane affinity conferred by the lipid anchor ( -6 kcal / mol , see below ) . in this study , we quantified how the interplay between lipid anchorage and electrostatics determines both membrane affinity and the range of allowed transducin orientations on the membrane surface . these properties change dramatically upon the activation - dependent dissociation of transducin into gt and g11 and are likely to significantly affect the protein - protein interactions and the subcellular localization of the transducin heterotrimer and its individual subunits . interactions of gt with the membrane and their implication for transducin activation it is well established that the membrane affinity of gt is determined predominantly by its lipid anchors ( 4 ) : the heterogeneous n - terminal acylation of gt ( 13 , 55 - 58 ) and the c - terminal farnesylation of g1 ( 59 , 60 ) . the majority of gt subunits are acylated by c12 or c14:2 lipids , which contribute approximately -6 kcal / mol to membrane binding ( 61 ) . farnesylation was shown to confer a membrane affinity comparable with that of a lauryl ( c12 ) anchor ( 62 ) and therefore can also add approximately -6 kcal / mol to membrane binding . in a maximal estimate , the contributions of both anchors are additive and sum to approximately -12 kcal / mol . our results show that under physiological conditions the electrostatic contribution to the membrane affinity of the heterotrimer is insignificant ( -0.3 kcal / mol ) , and therefore its membrane attachment is likely to be set exclusively by the lipid anchors . under hypotonic conditions , however , strong electrostatic repulsion counteracts the lipid anchors of gt ( fig . 5 ) and facilitates its release from membranes , as has been successfully utilized in several transducin purification protocols ( 26 - 29 ) . global orientation sampling enabled us to compare the probabilities of all previously proposed gt orientations on the membrane surface . the tilted orientations suggested by hessel et al . and chabre and le maire ( 24 , 25 ) are similar to the gt orientation identified by our analysis as the most favorable . on the other hand , parallel orientations ( 16 - 23 ) place the negatively charged patches around the c terminus of gt close to the negatively charged bilayer , making these orientations less favorable by 2 kcal / mol ( 30-fold difference in occupancy probability ) . although the magnitude of these energetic differences is relatively small , they are consistent with the dramatic charge distribution on gt , where most of the surface of the heterotrimer is negatively charged except for the small area around the lipid anchor attachment points ( fig . furthermore , we observe that in the electrostatically favorable orientations of gt , the gt - bilayer interface is relatively small ( e.g. fig . this small , positively charged gtfootprint on the membrane is consistent with the results of hessel et al . ( 24 ) , who showed that only a small number of negatively charged lipids directly interact with membrane - bound gt . they speculated that such a small interface might contribute to the fast diffusion of gt along the crowded membrane surface of rod outer segment disks , which is necessary for the rapid activation of many gt molecules by each rhodopsin ( see discussion in ref . 2 and see below ) . notably , these results may appear to contradict the cryo - em structure of membrane - bound gt ( 63 ) , which suggested a parallel gt orientation with a larger membrane interface and a close contact between the c terminus of gt and the membrane surface . however , the membranes used in that study contained 20% cationic lipids , which presumably attracted the negatively charged patches around the c terminus of gt. our calculations predict that these very patches are repelled by negatively charged biological membranes . our analysis also shows that electrostatic repulsion by the membrane is one of the factors limiting the rotational degrees of freedom of the heterotrimer . interestingly , all gt orientations that are lipid - allowed and electrostatically favorable are sterically predisposed to interact with rhodopsin ( supplemental fig . this suggests that the orientational confinement of transducin by lipid anchorage and electrostatics may contribute to its extremely rapid activation rate . indeed , the rate of transducin activation on the surface of photoreceptor membranes can reach several hundred gt molecules per photoexcited rhodopsin / s ( 2 , 6 , 7 ) , whereas in detergent solution the maximal speed of gt activation is only 30 - 50 gt molecules per second ( 9 , 64 ) . the faster gt activation rate in native membranes was attributed by ernst et al . ( 9 ) to the orientation of gt at the membrane surface before it encounters rhodopsin . in agreement with this hypothesis and , assuming that our calculated reduction in rotational entropy ( 1.8 kcal / mol ) lowers the activation energy for gt - rhodopsin complex formation by a similar value , this orientation confinement would accelerate gt activation by 20-fold . it should be noted that although our calculations were performed with a membrane bilayer , a significant portion of the photoreceptor membrane surface is occupied by rhodopsin . therefore , any direct projection of our analysis to transducin activation in vivo should be treated cautiously . quantification of the effect of gt orientation by the membrane on activation kinetics in vivo would require a more detailed understanding of the surface characteristics of native photoreceptor membranes and remains beyond the scope of this study . nevertheless , the ability of a single photoexcited rhodopsin to activate hundreds of gt molecules / s requires rapid diffusion of gt along the membrane bilayer ( 2 , 6 , 7 , 65 ) . therefore , any interactions between gt and nonactivated rhodopsin are expected to be weak enough not to impede this fast lateral diffusion . the orientation of transducin in relation to the membrane was also debated in the context of whether transducin binds to a monomeric or dimeric rhodopsin form ( ref . 66 for the most recent review on g protein - coupled receptor oligomerization ) . it has been suggested that monomeric rhodopsin can bind transducin in a tilted orientation ( 25 ) , whereas dimeric rhodopsin was modeled in a complex with transducin in a parallel orientation ( 67 ) . the tilted orientation we find would appear to be more consistent with monomeric rhodopsin . however , the energy differences we calculate are not large enough to preclude a reorientation of transducin so as to optimize its interaction with a rhodopsin dimer . membrane interactions of dissociated g11 and gtour analysis indicates that the electrostatic properties of gt and g11 are different from one another and from those of gt . in the case of g11 , ( 30 ) , who described a significant electrostatic attraction of g11 to the membrane , despite its negative net charge of -12 . because the averaged electrostatic affinity of gb11 to the membrane is the same with or without the constraints imposed by the farnesyl anchor , we consider these two energetic contributions as independent and additive . we estimate that electrostatics increase the membrane affinity of g11 from approximately -6 kcal / mol ( because of the farnesyl anchor alone ) to approximately -9 kcal / mol . in comparison with gt , g11 can occupy a wider range of orientations that are both lipid - allowed and electrostatically favorable ( fig . interestingly , the sites on g subunits that interact with different effectors are distributed over much of the g surface ( 68 ) , and therefore this wide range of allowed g orientations relative to the membrane can facilitate productive interaction with various effectors . calculations of gt interactions with the membrane are not as straightforward as those of g11 because the lipidated n terminus was proteolytically removed in all available gt structures . although we can not rule out the possibility that the n terminus of monomeric gt is intrinsically unstructured , previous studies of its close homolog gi1 showed that although its n terminus is present as an extended helix in the heterotrimer , in the monomer it packs against the rest of the subunit in a compact conformation ( 43 ) . a highly ordered and compact conformation of the myristoylated n terminus of gi1 was also shown in solution , and it was suggested that this property is conserved in homologous g subunits ( 44 ) . on the basis of this evidence , we assumed in our calculations that , following dissociation from g11 , the extended n - terminal helix of gt undergoes a similar conformational rearrangement . although gt and g11 have nearly identical net charges ( -13 and -12 , respectively ) , the distributions of negative charges on their surfaces are different . 3b ) , and our calculations predict that , unlike g11 , gt is repelled by the membrane in all lipid - allowed orientations . this reduces the membrane affinity of the compact gt conformation from the -6 kcal / mol provided by c12 or c14:2 lipid anchors to less than -4 kcal / mol . similarly , the membrane affinity of the minority of gt subunits that are modified by c14:1 or c14 lipids ( providing -7or -8 kcal / mol membrane affinity , respectively ) is reduced to less than -5or -6 kcal / mol . the membrane repulsion of gt could be lower if its n terminus is unstructured , or higher if the n - terminal linker of gt to its lipid anchor is not fully extended and flexible as we assumed . unlike gt and g11 , the linker length does matter for gt. a shorter or less flexible linker will increase gt repulsion by the membrane because the least repulsive orientation of gt falls right at the edge of the lipid - allowed region , where the electrostatic repulsion increases steeply ( fig . 3a ) . the repulsive gt-membrane interactions that we predict are consistent with the following experimental evidence . 1 ) gt binding to negatively charged membranes is significantly weaker than the binding of g11 ( 4 , 5 , 31 ) , despite the similar hydrophobicity of their lipid anchors . 2 ) g11 binding to nonmyristoylated gt lowers g11 affinity to negatively charged membranes ( 4 ) . 3 ) acidic ph facilitates the binding of gt to negatively charged membranes , which is non - detectable at neutral ph ( 5 ) . one of the properties of transducin that has attracted much attention in the past 5 years is its ability to undergo reversible , light - induced translocation from the rod outer segment to other subcellular compartments ( 10 , 69 - 72 ) . this massive translocation is thought to contribute to photoreceptor light adaptation and survival ( reviewed in refs . gt and g11 translocate apart from one another , and up to 90% of gt and 80% g11 move out of the rod outer segment within minutes ( 10 ) . the consensus is that transducin translocation in the light - induced direction requires subunit dissociation and is accomplished by diffusion ( 14 , 15 , 72 , 75 ) . surprisingly , the rate of this diffusion is comparable with that of soluble green fluorescent protein through the same compartment ( 75 ) , despite the fact that lipidated gt and g11 have to pass through narrow cytosolic spaces between hundreds of tightly packed membranous disks . our results suggest that the efficient translocation of monomeric gt may be facilitated by electrostatic repulsion , which is expected to reduce the affinity of gt to membranes . this may both accelerate the detachment of gt from the membrane and enable faster gt diffusion through the rod outer segment with minimal retardation by the disks . as would be expected , the translocation of the majority of gt subunits , which are modified by c12 or c14:2 lipids , is more pronounced than the translocation of the minority of gt subunits modified by slightly more hydrophobic lipids c14:1 and c14 ( 13 ) . in the case of g11 , its electrostatic properties enhance membrane binding , which is expected to impede translocation . accordingly , it has been shown that the efficient translocation of g11 requires phosducin ( 76 ) , a protein that stabilizes the binding of the farnesyl moiety inside a cleft within g1 ( 77 ) and neutralizes the electrostatic attraction of g11 to the membrane ( 30 ) . our results also reflect on how soluble transducin could reattach to the membrane from the cytosol , as occurs in rods recovering from illumination . although it remains to be determined experimentally whether transducin reattaches to the membrane in a heterotrimeric or in a dissociated state ( 14 ) , our results suggest the following . 1 ) the electrostatically favorable orientations of gt enable it to approach the membrane without experiencing an electrostatic energy barrier ( fig . 2 ) soluble g11 is actively attracted to the membrane in many orientations ( fig . 3 ) electrostatic interactions orient the lipid anchors of soluble gt or g11 toward the membrane , thereby favoring lipid anchor insertion into the membrane for both gt and g11.4)incontrast , electrostatic interactions oppose the approach of soluble gt to the membrane in orientations that enable membrane insertion of its lipid anchor ( fig . 78 that association with g11 is required to efficiently reattach gt to membranes . implications for membrane interactions of other g proteins our calculations were performed on bovine gt , but they are also applicable to all mammalian gt orthologs because they are > 98% identical in sequence . gt orthologs in more distantly related vertebrates also show > 90% sequence identity , both globally and when considering only their charged residues . similarly , murray et al . ( 30 ) showed that the electrostatic properties of not just g11 from different species , but all mammalian g subunits ( which share > 80% sequence identity ) , are generally conserved and resemble those of g11 . visualization of the gi112 heterotrimer ( pdb code 1gp2 ) in a similar orientation to that of gt in fig . the subunits are shown in connolly surface representation and colored pink ( ) , light green ( ) , and light blue ( ) . the conserved negatively charged patches discussed in the text are marked with red ( g12 ) and orange ( gi1 ) arrows . visualization of the gi112 heterotrimer ( pdb code 1gp2 ) in a similar orientation to that of gt in fig . the subunits are shown in connolly surface representation and colored pink ( ) , light green ( ) , and light blue ( ) . the conserved negatively charged patches discussed in the text are marked with red ( g12 ) and orange ( gi1 ) arrows . other members of the gi subunit subfamily ( go1 , go2 , gi1 , gi2 , and gi3 ) share a lower sequence identity with gt , ranging between 60 and 70% . nevertheless , the electrostatic potential maps of these related g subunits are similar , and in particular the dominant negative patches that produce gt-membrane repulsion are also observed in these proteins ( 32 ) . these similarities suggest that the electrostatic repulsion of dissociated g subunits by the membrane is common among all members of the gi subfamily . additionally , when we compared the electrostatic potential map of the gi112 heterotrimer ( 79 ) ( fig . in particular , the negatively charged patches that limit the rotation of gt are also present in the gi112 heterotrimer ( marked with arrows in fig . therefore , the characteristics of the dynamic interactions of the gt heterotrimer with the membrane are expected to apply to the entire gi subfamily . unlike gi subfamily members , g subunits that are palmitoylated only ( e.g. gq , gs , and g12 ) contain prominent basic patches at their n termini ( 32 ) . a number of these proteins have been shown to undergo cycles of reversible membrane association and dissociation , mediated by the cleavage and re - attachment of their labile palmitate anchors ( 11 , 80 , 81 ) . the basic electrostatic motif in these g subunits may initiate membrane binding ( 32 ) , whereas their subsequent palmitoylation would strengthen this attachment ( 80 , 81 ) . this interdependence , which could be viewed as an example of the two - signal model of membrane binding ( 80 , 81 ) , was recently observed in experiments analyzing the roles of the basic n - terminal motif and palmitoylation in membrane attachment of gq , gs , g14 , and g16 ( 82 , 83 ) . these results support the hypothesis that , unlike gt and its homologs , the subunits of the gq , gs , and g12 subfamilies are attracted to the membrane . in a broader context , it is well established that both membrane binding and subcellular localization of numerous peripheral membrane proteins are determined by various combinations of lipid anchorage and electrostatic interactions ( 30 , 33 - 40 ) . although g proteins follow this general theme , they also represent an example of how the activity - dependent dissociation of a multisubunit protein complex changes the interplay between lipid anchors and electrostatics . the unique electrostatic properties of the heterotrimer and its subunits thereby provide different membrane binding affinities and contribute to the specificity of their protein - protein interactions . therefore , extending the computational approaches used here to analyzing differences among a broad range of g proteins is a promising direction of future work .

the purpose of this report is to present apert syndrome patient by highlighting craniofacial characteristics and orthodontic approach to treatment.the patient , a 16-day - old female and the second child of healthy parents , was admitted to our department with primary complaint of cleft palate . she had a cone - shaped calvarium , midface hypoplasia , syndactyly of the hands and feet , hypertelorism , proptosis and cleft palate . after taking maxillary impression , an acrylic appliance was applied to orientate the growing and enable the feeding.a case with apert syndrome undergoes the orthodontic treatment for a long time and also a multidisciplinary approach is essential to determine the best collaborative corrective plan for the deficiencies of the patient . the heterotrimeric g protein transducin is a key component of the vertebrate phototransduction cascade . transducin is peripherally attached to membranes of the rod outer segment , where it interacts with other proteins at the membrane - cytosol interface . however , upon sustained activation by light , the dissociated gt and g11 subunits of transducin translocate from the outer segment to other parts of the rod cell . here we used a computational approach to analyze the interaction strength of transducin and its subunits with acidic lipid bilayers , as well as the range of orientations that they are allowed to occupy on the membrane surface . our results suggest that the combined constraints of electrostatics and lipid anchors substantially limit the rotational degrees of freedom of the membrane - bound transducin heterotrimer . this may contribute to a faster transducin activation rate by accelerating transducin - rhodopsin complex formation . notably , the membrane interactions of the dissociated transducin subunits are very different from those of the heterotrimer . as shown previously , g11 experiences significant attractive interactions with negatively charged membranes , whereas our new results suggest that gt is electrostatically repelled by such membranes . we suggest that this repulsion could facilitate the membrane dissociation and intracellular translocation of gt. moreover , based on similarities in sequence and electrostatic properties , we propose that the properties described for transducin are common to its homologs within the gi subfamily . in a broader view , this work exemplifies how the activity - dependent association and dissociation of a g protein can change both the affinity for membranes and the range of allowed orientations , thereby modulating g protein function .

, it accounts for the largest number of deaths from malignant neoplasms in women and the third largest number in men . however , since the recurrence rate of t1 colorectal cancer ( invasion no deeper than the submucosa ) without lymph node metastasis is approximately 1% , the outcome of t1 colorectal cancer is considered to be good . on the other hand , the incidence of synchronous liver metastases in colorectal cancer has been reported to be about 10% , yet t1 colorectal cancer with synchronous liver metastasis is considered to be rare [ 3 , 4 ] . therefore , there have been many reports of lymph node metastases , but few reports of the risk factors of synchronous or metachronous distant metastases from t1 colorectal cancer . we report a case of t1 colorectal cancer of the ascending colon with synchronous liver metastasis . the patient was a 68-year - old japanese man who was admitted to our hospital with a liver tumor that had been detected by abdominal ultrasonography . laboratory tests showed : erythrocyte count 484 10/mm ( normal 420554 ) , hemoglobin 15.9 g / dl ( normal 13.816.6 ) , leukocyte count 8,600/mm ( normal 3,5009,000 ) , platelet count 21.8 10/mm ( normal 15.536.5 ) , serum total protein 6.5 g / dl ( normal 6.38.1 ) , total bilirubin 0.69 mg / dl ( normal 0.31.2 ) , aspartate aminotransferase 28 iu / l ( normal 436 ) , alkaline phosphatase 224 iu / l ( normal 115359 ) , -glutamyl transpeptidase 53 iu / l ( normal 468 ) , blood urea nitrogen 16 mg / dl ( normal 921 ) , and creatinine 0.92 mg / dl ( normal 0.601.20 ) . carcinoembryonic antigen and ca19 - 9 were both elevated at 23.9 ng / ml ( normal 03 ) and 51 u / ml ( normal 037 ) , respectively . abdominal ultrasonography revealed a hyperechoic mass in segment 7 of the liver , 30 mm in diameter , with a circumferential hypoechoic area . abdominal computed tomography also showed mild enhancement of a liver tumor 30 mm in diameter at the portal phase ( fig . 1a ) . on colonoscopy , a pedunculated tumor with a central depression ( 20 mm in diameter ) was observed in the ascending colon , and this tumor was considered to be invading deeply into the submucosal layer ( fig . abdominal magnetic resonance imaging showed the liver tumor as a low - intensity mass in the t1 emphasis phase ( fig . 1c ) , as a high - intensity mass in the t2 emphasis phase ( fig . 1d ) and as a strongly high - intensity mass by administering the contrast medium ferumoxide ( fig . t1 colorectal carcinoma with liver metastasis was suspected , and right hemicolectomy with d3 lymphadenectomy and partial hepatectomy were performed simultaneously ( fig . the tumor was a well - differentiated tubular adenocarcinoma with 3,000 m invasion of the submucosal layer ( fig . 2c , the liver tumor showed histological findings similar to those of the primary colorectal carcinoma ( fig . the pathological stage according to the 7th edition of the tnm classification was stage iv ( t1n0m1 ) . his general condition worsened , and the patient ultimately died 14 months after the operation . a multicenter study in japan reported that 153 ( 8.5% ) of 1,806 patients with t1 colorectal cancer had lymph node metastases and 40 ( 2.2% ) of 1,806 patients had synchronous or metachronous distant metastases . among them , furthermore , when the focus was restricted to synchronous liver metastases , the incidence was 0.2% . also reported that the incidence of liver metastases from t1 colorectal cancer was 3.3% and that of synchronous liver metastases 0.9% . thus , the form of metastases from t1 colorectal cancer is mainly lymph node metastases . therefore , to date , there have been many reports of lymph node metastases but few reports of distant metastases . with respect to the risk factors of lymph node metastasis in t1 colorectal cancer , sakuragi et al . reported that the depth of submucosal invasion ( 2,000 m ) and lymphatic invasion significantly predicted the risk of lymph node metastasis in multivariate analysis . in addition , the depth of submucosal invasion ( 1,000 m ) , lymphovascular invasion , histological grade [ 7 , 8 ] and budding at the invasive front of the tumor [ 7 , 8 ] were thought to be significantly associated with lymph node metastasis . on the other hand , with respect to the risk factors of synchronous or metachronous distant metastases from t1 colorectal cancer , nodal metastases , depth of invasion and venous invasion are considered to be risk factors . these risk factors of distant metastasis are often common to those of lymph node metastases . it is considered that the highly malignant potential of t1 colorectal cancer , which can invade the submucosal layer broadly or exhibit poor differentiation , allows the tumor to invade the venous plexus of the submucosal layer and then develop distant metastases . in our case , submucosal invasion ( 3,000 m ) and venous invasion were recognized as risk factors . consequently , this case had multiple risk factors of distant metastasis . it has also been reported that the 5-year survival rate after liver resection in patients with synchronous liver metastases is 1938% , and that the prognosis of patients with resectable synchronous liver metastases is better than that of those with non - resectable liver metastases . however , even after curative resection , some cases have a poor prognosis for recurrence in the remnant liver and exhibit extrahepatic recurrence . in our case , postoperative adjuvant chemotherapy was undertaken for 6 months , however the prognosis was poor because of extrahepatic recurrence 9 months after the operation . hayashi et al . reported that extrahepatic recurrence was a prognostic factor after curative liver resection and noted that postoperative adjuvant systemic chemotherapy is necessary for patients with synchronous liver metastases because liver metastases should be considered equivalent to systemic disease . moreover , expert consensus statements by the american hepato - pancreato - biliary association also concluded that adjuvant systemic chemotherapy after curative liver resection must be supported because most patients with synchronous liver metastases concurrently exhibit extrahepatic metastases . however , the regimens and periods of postoperative adjuvant systemic chemotherapy were not fixed because of the absence of consensus for adjuvant chemotherapy after curative liver resection . there are few well - organized reports because t1 colorectal cancer with synchronous liver metastasis is extremely rare . on the other hand , the prognosis is often poor in patients with metachronous liver metastases after surgery for t1 colorectal cancer [ 13 , 14 , 15 ] . therefore , it is predictable that the prognosis of patients with synchronous liver metastasis is also poor . it is necessary to establish postoperative surveillance and a plan for adjuvant chemotherapy for t1 colorectal cancer with synchronous liver metastasis . we consider this case to be rare , and it is necessary to accumulate more cases to further elucidate the risk factors for liver metastasis from t1 colorectal cancer . the cases were collected from our melanoma database , and 30 patients who were treated from january 2001 to december 2010 were retrospectively reviewed . medical records were reviewed for demographics , clinical and pathological information , immediate as well as remote postoperative events and outcomes , with a minimal follow - up of 2 years or death . all patients were advanced stage ( american joint committee on cancer [ ajcc ] stage ii and iii ) melanoma patients , and for the operative treatment , we performed wide marginal surgery , which sometimes involved removal of the bone and surrounding tissue . and without considering the stage of the melanoma , we routinely performed sentinel lymph node dissection in all patients . therefore , we performed amputation with lymph node dissection for all patients who had melanoma of ajcc stage iii ( table 1 ) . after the surgery , we monitored patients for recurrence and metastasis every 3 months for the first 2 years and then gradually lengthened the interval between visits to yearly by the fifth year if no relapse of disease was observed . patients underwent history , physical examination , chest radiography , and radiography of the affected region at each visit . computed tomography , magnetic resonance imaging , and whole - body bone scans were obtained selectively , depending on whether there was evidence from the history , physical examination , and radiographs to warrant further study . data extracted included early diagnosis , sex , location of the tumor ( hand or foot ) , and for the clinical evaluation , we used the ajcc classification.10 ) also , patient 's mortality , follow - up duration , the date of the first operation , and metastasis to the sentinel lymph node were assessed . data on survival ( from the date of diagnosis to the date of last follow - up or death ) for the survival analysis was acquired from our database . for the statistical analysis , the kaplan - meier product was used to estimate curves for survival rate , and the log - rank test was used to evaluate differences between the survival curves . patients who were lost to follow - up or who were alive at the time of the last follow - up were censored at the date of their last follow - up . univariate t - test analysis was used to calculate the relative risk of each parameter . chicago , il , usa ) to perform statistical calculations and calculated confidence intervals of 95% for the statistical parameters . eighteen of the 30 patients ( 60% ) were men and 12 patients ( 40% ) were women , for a male - to - female ratio of 1.5:1 . most were diagnosed while in the sixth or seventh decade of life , with a mean age of 58.7 years ( range , 35 to 78 years ) . all of the patients had ajcc stage ii or iii disease at initial examination , and lesions were located predominantly on the sole and thumb ( fig . thirty patients who were diagnosed with high - grade malignant melanoma , underwent amputation of the tumor - involving extremity . the overall survival of patients after amputation of the malignant melanoma was 93% at 1 year , 76% at 2 years , and 67% at 5 years ( fig . fourteen of 30 patients were alive without disease , and six patients were dead with the disease at the end of the study ( table 2 ) . on the univariate analysis , diagnosis when over 70 years of age , tumor located on the lower extremity , and postoperative lymph node metastasis were found to predict poorer survival ; and this result was statistically significant ( p < 0.05 ) ( table 3 ) . and on the kaplan - meier analysis , patient 's gender and location of the tumor did not affect long term survival , whereas age at diagnosis , ajcc stage , and postoperative lymph node metastasis did impact the survival ( p < 0.05 ) ( table 4 ) . the overall survival curve was plotted according to age , sex , location , ajcc stage , and postoperative lymph node metastasis ( fig . as mentioned above , there were significant differences in early diagnosis , ajcc stage , and postoperative lymph node metastasis of malignant melanoma ( p < 0.05 ) . we also found amputation with aggressive lymph node dissection improved the 5-year overall survival of ajcc stage iii patients , with a survival rate of up to 32.1% . eighteen of the 30 patients ( 60% ) were men and 12 patients ( 40% ) were women , for a male - to - female ratio of 1.5:1 . most were diagnosed while in the sixth or seventh decade of life , with a mean age of 58.7 years ( range , 35 to 78 years ) . all of the patients had ajcc stage ii or iii disease at initial examination , and lesions were located predominantly on the sole and thumb ( fig . thirty patients who were diagnosed with high - grade malignant melanoma , underwent amputation of the tumor - involving extremity . the overall survival of patients after amputation of the malignant melanoma was 93% at 1 year , 76% at 2 years , and 67% at 5 years ( fig . fourteen of 30 patients were alive without disease , and six patients were dead with the disease at the end of the study ( table 2 ) . on the univariate analysis , diagnosis when over 70 years of age , tumor located on the lower extremity , and postoperative lymph node metastasis were found to predict poorer survival ; and this result was statistically significant ( p < 0.05 ) ( table 3 ) . and on the kaplan - meier analysis , patient 's gender and location of the tumor did not affect long term survival , whereas age at diagnosis , ajcc stage , and postoperative lymph node metastasis did impact the survival ( p < 0.05 ) ( table 4 ) . the overall survival curve was plotted according to age , sex , location , ajcc stage , and postoperative lymph node metastasis ( fig . as mentioned above , there were significant differences in early diagnosis , ajcc stage , and postoperative lymph node metastasis of malignant melanoma ( p < 0.05 ) . we also found amputation with aggressive lymph node dissection improved the 5-year overall survival of ajcc stage iii patients , with a survival rate of up to 32.1% . malignant melanoma is a relatively rare occurrence in the asian population compared to fair - skinned populations . limited data are available on skin cancer in asians , including information on the prevalence rates of malignant melanoma in different asian populations.11 ) in korea , the incidence of malignant melanoma increased from 1.02% of all skin malignancies in the 1980s to 15.6% in the 1990s.12,13 ) the mean age of diagnosis of melanoma was 58.7 years in our study , which was similar to other studies.14 ) in asian populations , malignant melanoma tends to occur 1.5 times more often in males than females , as in our study.14 ) unlike other studies , all of our patients were consulted from the dermatologic department with more than ajcc stage ii , which is advanced stage malignant melaoma . the difficulty in making the diagnosis of these malignant melanomas and the subsequent delay in proper treatment are factors likely to contribute to the preponderance of advanced lesions seen at the initial presentation.15 - 17 ) in this study , all of the patients presented with either ajcc stage ii or iii , and all cases were more than 1.5 mm thick . these factors ( stage , thickness , and level of invasion ) are important prognostic parameters in patients with advanced stage melanoma.18 - 21 ) after diagnosis of the melanoma , the affected digit must be amputated promptly . confirming a previous report,17 ) most studies of subungual melanoma have used amputation with or without lymph node dissection , yielding 5-year survival rates of 16% to 32%,22,23 ) whereas others using amputation with lymph node dissection or regional limb perfusion have reported 5-year survival rates of 25% to 32% , with minimal morbidity and no mortality.24,25 ) in addition to amputation although the use of these treatment modalities did not affect patient survival , perfusion may reduce the incidence of local disease recurrence . there is still controversy about the significance of the anatomical site of origin in prognosis . in our study , 85% of the melanomas were located on the acral area ( palm , sole , and subungal area ) . however , when compared with melanomas arising in other locations , sex and thickness of tumor were not significantly different . according to the location of the tumor , our institution performed wide marginal resection of the tumor , that is , amputation . but , treatment of melanomas on the hands and feet is particularly challenging for surgeons due to the functional uses of these body parts and the difficulty of obtaining the conventionally recommended margins . however , in cases of advanced stage melanoma as in our studies , amputation is usually required due to the paucity of soft tissue between the tumor and the bone . therefore , although amputation is not commonly recommended in general , it should be the first recommendation in cases of advanced stage melanomas that have insufficient safety margins . balch et al.26 ) reported 5-year survival rates of 13% to 37% in patients who showed lymph node metastasis at the time of diagnosis . although , in our study , patients who were diagnosed as ajcc stage iii showed a poorer survival rate than stage ii patients , with aggressive lymph node dissection during the amputation , we found an improved survival rate of ajcc stage iii patients of up to 32.1% , higher than in other reports ( table 5).27 - 30 ) in korean melanoma patients , for the treatment of high grade melanomas on the extremities after amputation , early diagnosis and postoperative follow - up for evaluation of lymph node metastasis are critical factors for long term survival . by performing lymph node dissection during amputation , we may improve the survival rate in advanced stage melanoma patients .

the patient was a 68-year - old man who was admitted to our hospital with a liver tumor . abdominal ultrasonography and computed tomography revealed a liver tumor 30 mm in diameter . on colonoscopy , a pedunculated tumor with a central depression ( 20 mm in diameter ) was observed in the ascending colon , and this tumor was considered to be invading deeply into the submucosal layer . right hemicolectomy with d3 lymphadenectomy and partial hepatectomy were performed simultaneously . on histopathological examination of the resected specimen , the tumor was a well - differentiated tubular adenocarcinoma with 3,000 m invasion of the submucosal layer . the liver tumor showed histological findings similar to those of the primary colorectal carcinoma . the pathological stage according to the 7th edition of the tnm classification was stage iv ( t1n0m1 ) . nine months after the operation , computed tomography revealed hepatic hilar lymph node metastases and a great deal of ascites . the patient ultimately died 14 months after the operation . backgrounda retrospective study was conducted to review the overall survival and treatment outcomes of high grade melanoma in the extremity to explore the clinical features of malignant melanoma of the hand and foot , and the therapeutic efficacies and survival rate after amputation.methodsthe clinical data of 30 patients with malignant melanoma of the hand and foot ( confirmed by pathological examination ) , who were admitted and treated in our hospital between 2001 and 2010 , were analyzed retrospectively . we analyzed variables affecting overall and disease - free survival.resultsthirty patients ( 18 men and 12 women ) treated with an amputation procedure for malignant melanoma in the hand or foot constituted the study cohort . the average age of the patients at the time of diagnosis was 58.7 years . univariate analysis for overall melanoma survival revealed that diagnosis at over 70 years of age , postoperative lymph node metastasis , and location of the tumor were significant prognostic factors . and on the kaplan - meier survival curve , old age , american joint committee on cancer stage and postoperative lymph node metastasis showed statistically significant differences in the 5-year survival rate . also , amputation with aggressive lymph node dissection showed improved long term survival in advanced stage melanoma.conclusionsin korean melanoma patients , for the treatment of high grade melanomas in the extremities after amputation , early diagnosis and postoperative follow - up for evaluation of lymph node metastasis are critical factors for long - term survival . and by performing lymph node dissection during amputation , we may improve the survival rate in advanced stage melanoma patients .

granulocyte macrophage colony - stimulating factor ( gm - csf ) is a hematopoietic growth factor which was originally recognized as a stimulator for the proliferation of granulocytes and macrophages from bone marrow precursor cells . it has also been shown to promote the survival and activation of mature myeloid cells and therefore contributes to the maintenance of innate immune homeostasis . recent studies suggest that gm - csf also has proinflammatory functions and plays critical roles in the development of autoimmune and inflammatory diseases , particularly in th17 driven diseases [ 3 , 4 ] . major sources of gm - csf include activated t and b cells , monocytes / macrophages , endothelial cells , fibroblasts , and other sources such as neutrophils , eosinophils , epithelial cells , mesothelial cells , chondrocytes , paneth cells , and tumor cells [ 57 ] . the production of gm - csf in t cells is stimulated by il-1 and il-23 in mice [ 3 , 8 ] , il-1 and il-12 in humans , and also prostaglandin e2 . in fibroblasts , endothelial cells , chondrocytes , and smooth muscle cells , it is stimulated by tnf- and il-1 , and in macrophage / monocytes it is stimulated by toll like receptors ( tlrs ) . in lymphocytes , the transcription factor nuclear factor of activated t cells ( nfat ) is reported to be required for the production of gm - csf [ 11 , 12 ] . however , the production of gm - csf can be inhibited by ifn- , il-4 , il-10 , and also pharmacological agents such as cyclosporine a [ 16 , 17 ] or glucocorticoids . the gm - csf receptor is expressed on myeloid cells and on some nonhaemopoietic cells such as endothelial cells but not on t cells [ 19 , 20 ] . the gm - csf receptor is a heterodimer of an -subunit which binds gm - csf with low affinity and a signaling c - subunit which is shared with the il-3 and il-5 receptors . the c - subunit constitutively associates with janus kinase 2 ( jak2 ) and is tyrosine phosphorylated by it resulting in an assembly of dodecameric signaling complex and initiation of signaling . the effects of gm - csf are mediated in a dose - dependent manner , by two -chain residues : ser585 and tyr577 . at low concentrations of gm - csf , as in normal healthy tissues , signaling occurs via ser585 of the -chain , which leads to activation of the pi-3 kinase pathway and results in myeloid cell survival . at high concentrations of gm - csf , as at the site of inflammation , signaling via ser585 is extinguished and signaling occurs exclusively via tyr577 residue , which activates the jak2/stat5 pathway , ras / mitogen - activated protein kinase pathway and pi-3 kinase pathway , resulting in cell survival , proliferation , and activation [ 2326 ] . gm - csf stimulates proliferation and activation of macrophages , monocytes , neutrophils , eosinophils , dendritic cells , and microglia [ 1 , 27 ] . however , since gm - csf - deficient mice did not have a defect in myeloid cell development , a redundant role of gm - csf in myeloid cell development and differentiation under steady state or homeostatic conditions is predicted . in addition to its function as a hematopoietic growth factor , gm - csf is now recognized to have a variety of functions on mature hemopoietic cells . gm - csf enhances proinflammatory cytokine production , antigen presentation [ 30 , 31 ] , and phagocytosis [ 3235 ] and promotes leukocyte chemotaxis and adhesion [ 5 , 3638 ] . gm - csf deficient mice have increased susceptibility to pulmonary [ 28 , 3941 ] and intestinal infections followed by systemic infection , indicating its importance in maintaining immune homeostasis particularly in the lung and intestines , which are constitutively exposed to pathogens . gm - csf stimulates the terminal differentiation of macrophages and the acquisition of normal immune functions via the transcription factor pu.1 . gm - csf also regulates phagocytosis of microbial pathogens by macrophages through the upregulation of pathogen associated molecular pattern ( pamp ) receptors such as c - type lectins including mannose receptors or dectin-1 , scavenger receptors , integrins , or fc receptors via pu.1 [ 24 , 3234 , 43 ] . complement - dependent phagocytosis is also enhanced by gm - csf to control microbial pathogens . gm - csf also upregulates the expression of tlr2 , tlr4 , or cd14 and boosts the production of proinflammatory cytokines such as tnf , il-6 , il-12p70 , il-23 , or il-1 [ 24 , 32 , 45 , 46 ] , leading to polarization of macrophages to the m1- ( classic- ) like phenotype , thus , promoting th1th17 responses [ 29 , 47 , 48 ] and contributing to tissue destruction . on the other hand , m - csf polarizes macrophages to the m2- ( alternative- ) like phenotype , which produces anti - inflammatory cytokines such as il-10 and cc - chemokine ligand 2 ( ccl2 ) and promotes tissue repair and remodeling . gm - csf also regulates many functions in macrophages including cell adhesion , pulmonary surfactant lipid and protein catabolism , and several important antimicrobial activities such as the production of reactive oxygen species ( ros ) or expression of antimicrobial enzymes . gm - csf positively regulates the development of migratory cd103cd11b dcs but negatively regulates the development of resident cd8 dcs . gm - csf also strongly induces the development of inflammatory monocyte - derived dcs ( modcs ) in vitro . however , it has not been well established whether gm - csf also regulates the development of modcs in vivo . it was reported that the number of modcs was increased in gm - csf transgenic mice . furthermore , nf-b1-dependent gm - csf production in cd4 t cells was reported to be required for the generation of modcs in inflammatory arthritis and antigen - induced peritonitis mouse models . the number of modcs was markedly reduced in draining lymph nodes from gm - csf/ mice with inflammatory arthritis or in the spleen of mice reconstituted with nf-b1/ cd4 t cells in acute peritonitis , demonstrating that gm - csf contributes to the differentiation of these cells during inflammation in vivo . on the other hand , gm - csf was shown to be dispensable for the differentiation of modcs , at least during acute infections , since the number of modcs was not decreased in gm - csf/ mice or gm - csf receptor deficient mice during acute infections [ 55 , 56 ] . these data indicate that although gm - csf strongly regulates the production of modcs in vitro and in vivo , there may be another gm - csf - independent pathway for the development of modcs . besides the regulation of dc development , gm - csf also upregulates cross - presentation , bacterial uptake , or production of proinflammatory cytokines such as il-6 or il-23 in resident dcs . in mature neutrophils , gm - csf upregulates the expression of the integrin cd11b , which increases cellular adhesion and tissue entry . gm - csf also upregulates the antimicrobial functions of neutrophils , such as phagocytosis or ros production . however , the expression of pu.1 in neutrophils of autoimmune pulmonary alveolar proteinosis patients was normal , indicating that gm - csf is not involved in neutrophil differentiation . among b cells , the innate - like b1 b cells reside predominantly in serosal cavities such as the pleural or peritoneal cavity . in response to microbial infection , b1a b cells ( a subset of b1 b cells ) recognize bacteria via direct tlr - dependent pathogen recognition and differentiate into innate response activator ( ira ) b cells , which produce gm - csf and also express the gm - csf receptor [ 59 , 60 ] . gm - csf acts on its receptor in an autocrine manner and induces igm production from b cells [ 59 , 61 ] . mixed chimeric mice with b cell - restricted gm - csf deficiency showed high bacterial titer and morbidity after infection but did not show alveolar proteinosis , indicating that b cell - derived gm - csf is necessary for protective igm responses but dispensable for surfactant clearance by alveolar macrophages . these data indicate that the cellular source and location of gm - csf is important . although gm - csf is widely expressed in both stromal and hematopoietic cells , recent murine studies suggest that gm - csf from cd4 t cells is essential in inflammatory mouse models such as experimental autoimmune encephalomyelitis ( eae ) , arthritis models such as collagen - induced arthritis ( cia ) or skg - arthritis , interstitial lung disease in skg mice ( skg - ild ) , peritonitis , or myocarditis [ 3 , 4 , 54 , 57 , 6264 ] . although gm - csf is known as one of the th17 cytokines , th1 cells and th2 cells also express gm - csf [ 6467 ] . moreover , recent studies represent the existence of gm - csf - producing th cells distinct from th1 , th2 , or th17 cells [ 62 , 64 ] ( figure 1 ) . th17 cells have been shown to be strong inducers of tissue inflammation and autoimmune diseases . however , a number of studies determined that il-17 inhibition does not prevent but rather only ameliorates the development of eae [ 3 , 4 , 68 , 69 ] , cia , skg - arthritis , or skg - ild and that neutralizing il-17 is a rather unsatisfactory method for blocking th17 mediated diseases [ 71 , 72 ] . recently , it was reported that classical th17 cells , which mainly produce il-17 , are not pathogenic and that th17 cells have high plasticity . th1/17 cells are characterized by their ability to coproduce il-17 , ifn- , and gm - csf and are identified by the coexpression of t - bet , rort , and the chemokine receptors cxcr3 and ccr6 [ 9 , 7478 ] . human th1/17 cells also express cd161 , a hallmark of th17 progeny cells in humans that is induced by rort [ 76 , 79 , 80 ] . recent studies report that gm - csf is critical for the pathogenicity of th17 cells [ 3 , 4 ] and the presence of th1/17 cells was observed at the inflammatory site of inflammatory bowel disease ( ibd ) , multiple sclerosis ( ms ) , and juvenile idiopathic arthritis ( jia ) [ 76 , 78 , 81 , 82 ] . in mice , il-23 and il-1 induce the production of gm - csf in t cells whereas il-12 suppresses its expression [ 3 , 4 , 77 , 83 ] . in contrast , in humans il-1 renders th17 cells sensitive to il-12 and both il-1 and il-12 promote the differentiation of th1/17 cells [ 9 , 75 , 81 , 84 , 85 ] ( figure 1 ) . as described in section 2.3 , gm - csf induces the differentiation of m1-like macrophages and upregulates the production of proinflammatory cytokines such as il-6 , il-12 , il-23 , or il-1 from antigen presenting cells ( apcs ) . this results in further differentiation of th17 and th1/17 cells , thus creating a positive feedback loop [ 3 , 63 ] . studies show that gm - csf expression in cd4 t cells is not regulated by t - bet [ 3 , 4 ] and ror - responsive elements are identified in the promoter of the gene encoding gm - csf . moreover , ectopic rort expression in cd4 t cells results in gm - csf production , indicating that gm - csf production in th1/17 cells is induced by rort . conversely , rort - deficient cd4 t cells can produce gm - csf , indicating the existence of additional pathways to induce gm - csf production in cd4 t cells . recently , it was reported that il-2- or il-7-activated stat5 promotes the generation of gm - csf - producing cd4 t cells with low or undetectable expression of t - bet , gata-3 , rort transcripts . these cells represent a new distinct subset of th cells , namely , th - gm [ 62 , 64 ] . in humans , these th - gm cells are identified as ccr10ccr4cxcr3ccr6 th cells . it was reported that il-7-activated stat5 directly bound to promoter regions of the gene encoding gm - csf . the contribution of il-7 has been implicated in autoimmune diseases such as multiple sclerosis or rheumatoid arthritis [ 86 , 87 ] , which also suggests the contribution of th - gm cells in these diseases . high expression of il-3 , which is also involved in several autoimmune diseases [ 8890 ] , was also reported in th - gm cells . these cells were reported to be able to induce a more severe experimental autoimmune encephalomyelitis ( eae ) than th17 or th1 cells [ 62 , 64 ] . it is possible that th - gm cells provide gm - csf to induce the expression of il-23 from apcs and cooperate with th1/17 or th1 cells to exacerbate the development of inflammation . it was reported that th1 cells also need gm - csf to mediate inflammation in the central nervous system ( cns ) . however , the amount of gm - csf produced by th1 cells is found to be consistently lower than that produced by th17 cells , particularly during in vitro culture . although a positive correlation was found between gata-3 cells and gm - csf cells in the nasal mucosa of patients with allergic rhinitis , there is no study to our knowledge that directly analyzes the role of gata-3 in gm - csf production . further investigation is therefore needed to elucidate the precise mechanism of gm - csf production from cd4 t cells and their contribution to the development of autoimmune and inflammatory diseases . multiple sclerosis ( ms ) is a chronic inflammatory disease of the central nervous system and is pathologically characterized by demyelination and subsequent axonal degeneration . past studies have shown that cd4 t cells play a critical role in the development of ms and experimental autoimmune encephalomyelitis ( eae ) , a widely used mouse model of ms . it has been widely believed that th17 cells are the main encephalitogenic population in autoimmune inflammation ; however , il-17 has been found to be dispensable for the development of eae [ 4 , 93 ] . on the other hand , gm - csf deficiency or neutralization of gm - csf has been reported to prevent the onset of eae [ 94 , 95 ] . the administration of recombinant gm - csf worsened the disease in eae and elevated concentrations of gm - csf have been reported in the cerebrospinal fluid but not in the serum of patients with relapsing - remitting or secondary progressive ms [ 96 , 97 ] . recent findings show that gm - csf is secreted by cns - infiltrating helper t cells and is essential for encephalitogenicity in eae [ 3 , 4 , 64 ] . gm - csf induces the proliferation and activation of microglial cells , which is required for the onset of eae [ 95 , 98 ] . activated microglial cells produce highly neurotoxic substances such as ros , nitrogen species , glutamate , and tnf- [ 99102 ] . furthermore , gm - csf - producing cd4 t cells induce the differentiation of neurotoxic m1-like phenotype of microglia and upregulate the production of proinflammatory mediators such as il-1 , il-6 , and tnf , which contribute to myelin sheath damage , via upregulation of tlr and cd14 expression . studies also show that gm - csf is required for recruitment of peripheral myeloid cells that contribute to blood - brain barrier ( bbb ) and blood - spinal cord barrier ( bscb ) disruption and demyelization into the cns [ 104 , 105 ] . as we described in section 3.1 , gm - csf induces the polarization of the m1-like macrophage phenotype and exacerbates the positive feedback loop of th17 and th1/17 differentiation . indeed , an increased m1/m2 profile ratio of monocyte / macrophages in the blood as well as in the cns favors relapsing eae and a reduced m1/m2 ratio promotes an attenuated manifestation of the disease . the ability of cns - invading myeloid cells to respond to cd4 t cell - derived gm - csf was shown to be vital for the development of eae . taken together , cns - infiltrating cd4 t cells initially activate microglia and induce production of proinflammatory cytokines , which contribute to myelin sheath damage . this initial neuroinflammation results in bbb destruction and leukocyte infiltration into the cns parenchyma , followed by restimulation of t cells by resident and infiltrating apcs , leading to further apc activation . these reports indicate that gm - csf plays a central role in ms and indicate that the inhibition of gm - csf will be a useful therapeutic strategy for ms . mor103 , a fully human monoclonal antibody that binds human gm - csf , is currently being tested in a phase ib trial for ms ( table 1 ) . rheumatoid arthritis ( ra ) is a systemic chronic autoimmune disease characterized by persistent and erosive inflammatory polyarthritis . recent studies indicate that gm - csf plays a central role in the pathogenesis of ra as in ms , by activating or promoting differentiation and survival of macrophages and neutrophils [ 109 , 110 ] . the concentrations of gm - csf were elevated in the synovial fluid and plasma of ra patients [ 111 , 112 ] . a case report showed that the administration of recombinant gm - csf exacerbated the disease activity of ra . the frequency of gm - csf - producing th cells was significantly increased in synovial fluid cells compared to peripheral blood mononuclear cells ( pbmcs ) in patients with juvenile idiopathic arthritis ( jia ) and correlated with erythrocyte sedimentation rate ( esr ) levels [ 81 , 114 ] . synovial gm - csf - producing t cells were predominantly cd161 positive and coexpressed ifn- but not il-17 , indicating that these cells are th1/17 cells . alternatively , human synovial fibroblasts and chondrocytes were also reported to produce gm - csf in response to il-1 and tnf stimulation [ 115 , 116 ] . the contribution of gm - csf in the development of arthritis was also reported in several mouse models of arthritis . in the collagen - induced arthritis ( cia ) model , gm - csf deficient mice failed to develop arthritis , and the administration of anti - gm - csf neutralizing antibodies ameliorated existing disease , prevented disease progression , and reduced the concentrations of tnf and il-1 in the joints of treated mice . on the other hand , gm - csf administration exacerbated arthritis in cia . in skg mice , another model of autoimmune arthritis , gm - csf , upregulated proinflammatory cytokine production such as il-1 or il-6 from macrophages in a dose dependent manner [ 63 , 120 ] . this in turn induced further differentiation and expansion of il-17-producing and gm - csf - producing cd4 t cells . the progression of arthritis in skg mice was inhibited by the neutralization of gm - csf and slightly by the neutralization of il-17a , indicating that gm - csf plays a more critical role than il-17a in skg arthritis . mavrilimumab , a fully human anti - gm - csf receptor antibody , is currently being developed and a phase ii study in ra patients reported significant efficacy with no serious adverse events such as pulmonary alveolar proteinosis . in this study of patients with active ra despite methotrexate treatment , 55.7% of all participants treated with mavrilimumab met the primary end point of achieving 1.2 decrease from baseline in the disease activity score ( das28-crp ) at week 12 . at the highest dose of mavrilimumab ( 100 mg ) , 66.7% of subjects met the primary end point versus only 34.7% of the subjects in the placebo group . mor103 , which is also being tested for ms , has shown preliminary evidence of efficacy in a phase ib / iia trial for patients with active ra . subjects receiving higher doses of mor103 ( 1.0 and 1.5 mg / kg ) showed significant improvement in das28 scores and joint counts and significantly higher european league against rheumatism response rates than subjects receiving placebo . both mavrilimumab and mor103 showed rapid treatment responses and provided evidence of clinical efficacy that support further clinical investigation . a list of ongoing / completed clinical trials targeting gm - csf or its receptor is presented in table 1 , with more information available at clinicaltrials.gov . pulmonary alveolar proteinosis ( pap ) is a rare syndrome characterized by the accumulation of surfactant in pulmonary alveoli resulting in varying degrees of respiratory insufficiency and myeloid cell dysfunction leading to increased risk of infection [ 123 , 124 ] . several clinical forms of pap have been identified including autoimmune pap caused by gm - csf autoantibodies , hereditary pap caused by gm - csf receptor mutations , and secondary pap associated with various underlying clinical disorders which is presumed to cause this syndrome by reducing alveolar macrophage numbers or function . it is also reported that gm - csf deficient mice develop abnormal lung histology that is virtually indistinguishable from human pap . pulmonary surfactant is tightly regulated by balanced production , secretion , reuptake , and catabolism within alveoli . gm - csf regulates surfactant catabolism in alveolar macrophages via pu.1 but does not regulate surfactant endocytosis or uptake and catabolism of surfactant by alveolar epithelial cells type ii ( aec - ii ) [ 32 , 127 ] . gm - csf is also required to stimulate numerous immune functions and terminal differentiation of alveolar macrophages and induction of igm production from b cells [ 60 , 61 ] . therefore , gm - csf deficient mice have high susceptibility to pulmonary infections [ 28 , 3941 ] accompanied by systemic infections . based on the pathogenesis of pap , several new therapeutic approaches for treating autoimmune pap targeting gm - csf are in clinical trials , including plasmapheresis , gm - csf administration [ 129 , 130 ] , and rituximab [ 131 , 132 ] . the mechanism of pulmonary fibrosis in interstitial lung disease ( ild ) has been studied using idiopathic pulmonary fibrosis ( ipf ) or a bleomycin - induced mouse model of pulmonary fibrosis . pulmonary fibrosis in ipf is considered to be inflammation - independent and mainly initiated by tgf- produced by damaged epithelial cells . in fact , anti - inflammatory therapies have little benefit in ipf [ 133135 ] . however , neutrophils , which produce ros , mmps , neutrophil elastase , or myeloperoxidase and cause lung parenchymal and stromal cell injury [ 136138 ] or th2 cytokines such as il-4 or il-13 , which induce fibroblast differentiation or extracellular matrix synthesis [ 139 , 140 ] , have been reported to contribute to lung fibrosis to some extent . additionally , gm - csf was reported to take part in the progress of pulmonary fibrosis . it was reported that tnf-induced endothelin-1 ( et-1 ) upregulates gm - csf production from airway smooth muscle cells ( asmcs ) and that gm - csf was increased in the bronchoalveolar lavage fluid ( balf ) of patients with pulmonary fibrosis [ 142 , 143 ] . gm - csf stimulates macrophages to release profibrotic cytokines and also induces fibrosis by direct stimulation of airway smooth muscle cells [ 144 , 145 ] . in fact , overexpression of gm - csf in the lungs led to severe neutrophil , eosinophil , and macrophage infiltration and fibrotic reactions [ 145147 ] . in contrast with idiopathic pulmonary fibrosis ( ipf ) , connective tissue disease - associated ild ( ctd - ild ) is often characterized by a clearer response to immunosuppression , indicating that autoimmune / inflammatory mechanisms play a more significant and central role in the pathogenesis of ctd - ild [ 148 , 149 ] . skg mice , a model of autoimmune arthritis , were reported to develop chronic - progressive interstitial lung disease ( ild ) that histologically resembles ctd - ild [ 63 , 150 , 151 ] . recently , it was reported that ild in this mouse was characterized by massive infiltration of th17 cells , gm - csf - producing cd4 t cells , and cd11bgr1 neutrophils with fibrosis . naive skg t cells were skewed to differentiate into gm - csf - producing cells . furthermore , gm - csf secreted by t cells enhanced il-6 and il-1 production by macrophages , which in turn enhanced differentiation of il-17a and/or gm - csf - producing t cells and infiltration of neutrophils into the lungs . neutralization of gm - csf completely blocked the development of this ild , whereas neutralization of il-17a did not , showing that gm - csf not il-17a is critical for the development of ild in skg mice . importantly , neutralization of gm - csf ameliorated ild in skg mice even after the onset of ild , suggesting that gm - csf inhibition is a useful therapeutic strategy for ctd - ild . mavrilimumab , a fully human anti - gm - csf receptor antibody and mor103 , a fully human monoclonal anti - gm - csf antibody , are undergoing clinical trials in ra patients [ 121 , 122 ] . recent developments suggest that the development of crohn 's disease ( cd ) is caused by a mucosal innate immunodeficiency with a variety of genetic defects and a dysfunction of granulocytes , macrophages , and intestinal epithelial cells [ 153 , 154 ] . in the intestine , gm - csf contributes to gut barrier function and resistance to bacterial translocation by promoting the recruitment and activation of monocytes / macrophages , neutrophils , and dcs . gm - csf also promotes tissue repair via increased intestinal epithelial cell proliferation and increased macrophages as effectors of wound healing [ 155157 ] . however , in cd patients , the inherent defects in the mucosal barrier increase the translocation of pathogens to the bowel tissue . moreover , increased levels of gm - csf autoantibodies have been reported in patients with ileal / ileocolonic cd , compared with those with colon involvement only , ulcerative colitis ( uc ) patients , or healthy controls . the high levels of gm - csf antibodies directly correlated with disease activity and inversely correlated with neutrophil phagocytic activity . increased gm - csf autoantibodies affect mucosal integrity , bacterial killing and neutrophil migration , proliferation , and survival . gm - csf deficient mice also developed a more severe intestinal and systemic infection after enteric infection and were more susceptible to acute dextran sodium sulfate- ( dss- ) induced colitis . both severity of infection and colitis were largely prevented by gm - csf administration [ 160 , 161 ] . on the other hand , gm - csf overexpression in the stomach leads to autoimmune gastritis and experimental peritonitis or intraperitoneal lps exposure in gm - csf deficient mice resulted in blunted proinflammatory responses and mortality . the expression of gm - csf at the mrna level increases from the stomach distally down through the colon , indicating that gastrointestinal expression of gm - csf expression parallels bacterial localization . these data indicate that the threshold of gm - csf levels between inflammation and immune homeostasis can vary with tissue location or organs . these findings indicate that administration of gm - csf could be beneficial for the treatment for cd patients . initial reports indicated that the administration of gm - csf in cd patients with moderate to severe disease activity had a high rate of clinical response and remission with minimal adverse effects [ 165 , 166 ] . moreover , a randomized phase ii clinical trial demonstrated that gm - csf was significantly more effective than placebo in obtaining a corticosteroid - free clinical remission . conversely , a recent large randomized trial found that it was no more effective than placebo for induction of clinical remission or improvement in active cd . cd patients are known to have substantial heterogeneity in pathogenic mechanisms and so gm - csf as a therapy may only be appropriate for a subgroup of patients . additionally , it is also important to verify the efficacy of gm - csf not only for the induction of remission but also for the maintenance of remission . therefore , further studies are needed to elucidate the efficacy of gm - csf as a treatment as well as the appropriate patient character or phase of disease to apply gm - csf administration as a therapy . gm - csf also was reported to take part in th2 response in allergic airway inflammation via activation of dcs [ 169171 ] . in mouse models of asthma , allergen - exposed epithelial cells release gm - csf which activates dcs and also prolongs eosinophil survival [ 170 , 172 ] . consequently , gm - csf neutralization reduced allergic hyperresponsiveness in mice models [ 169 , 170 , 172 ] . anti - gm - csf antibody , is tested in a phase ii trial for severe asthma ( table 1 ) . gm - csf plays pivotal roles not only in maintaining immune homeostasis but also in exacerbating inflammatory reactions . recent findings indicate that gm - csf inhibition will be an attractive therapeutic strategy for many autoimmune and inflammatory diseases . studies also indicate that it is necessary to monitor possible side effects such as pap or cd , although gm - csf inhibition has no demonstrated serious adverse reactions so far , which is indicative of its wide therapeutic index [ 63 , 173 , 174 ] . further studies are necessary to identify the molecular mechanisms that regulate gm - csf production and the role of gm - csf in the development of inflammatory diseases to devise preventive or curative strategies for autoimmune and inflammatory diseases . hypertrophic cardiomyopathy ( hcm ) is the most common cause of sudden death in young patients . whereas especially patients with a nonobstructive form of hcm are thought to stay stable for a long time ; a small distinct subset of patients can develop end stage heart failure , which includes a reduction of systolic function and a transition from a former hcmphenotype to a dilated phenotype.1 several mechanisms underlying this transition are discussed and may include specific genetic backgrounds and microischemiainduced damages . in addition , temporary progression of the disease can be induced because of not primarily cardiacdependent stress factors . therefore , the differential diagnosis of the socalled end stage hcm is important not to overlook specific treatment options . we report here a case from a young hcm patient where cardiac inflammatory but not primarily hcmdependent processes induced a decompensating dilated cardiomyopathy ( dcm)like phenotype and show that an antiinflammatory treatment led to a reverse remodelling under these conditions . we report about a 17yearold woman with known hcm without left ventricular ( lv ) outflow obstruction characterized by a concentric hypertrophy ( anteriorseptal wall measured 15 mm , posterior 16 mm ) , preserved lv ejection fraction ( ef ) , and a wolffparkinsonwhite ( wpw ) syndrome for which the patient was already ablated twice in her childhood . she was submitted to our department for evaluation of a progression of heart failure , indicated by a new diagnosed drop of the lvef to 40% , hypokinesia of the apical and inferior wall , an increase in lv diameters ( lv end diastolic diameter ( lvedd ) : 59 mm ; mitral valveseptum : 18 mm ) , increased filling pressures , and a mitral regurgitation grade ii . clinically , she complained of chest pain , dyspnea already at rest ( nyha iii ) , and paroxysmal palpitations with presyncopal events during the past 6 months . blood analysis showed increased nterminal probrain natriuretic peptide ( ntpbnp ) levels of 5201 pg / ml and slightly increased transaminases ( alt 40 u / l , ast 60 u / l ) , without changes in tnt , hscrp , or leukocyte numbers . over the following 7 days , based on these findings , we discussed at least four possible differential diagnosis : ( 1 ) tachycardiainduced cardiomyopathy ( cm ) , ( 2 ) progression of hcm including ( micro)angiopathyinduced changes , ( 3 ) development of a severe primary mitral valve regurgitation , and ( 4 ) another undetected acquired form of cm . in an invasive electrophysiological evaluation , no malignant heart rhythm disturbances were inducible . we found a sufficient working atrioventricular node without any signs of accessory bundles excluding a wpwsyndrome induced tachycardiomyopathy . for further evaluation of the myocardial structure , we performed cardiac magnetic resonance imaging ( mri ) . the mri showed a significant hypertrophic and dilated lv ( lv diastolic volume of 286 ml ) with a further reduced lvef ( 17% ) . there was a pronounced signal of late gadolinium enhancement ( lge ) sequences of myocardial necrosis ( scar ) and/or fibrosis to detect , especially in the septum , apical and lateral part of the lv ( reticular delayed enhancement ) ( figure 1 ) . further on , there were no signals of myocardial inflammatory processes in the t1weighted and t2weighted images detected . to investigate whether or not a primarily hcmdependent progression of the disease or other etiologies were responsible for these findings , we evaluated right ventricular endomyocardial biopsies ( embs ) after exclusion of coronary abnormalities . histological characterization of the embs demonstrated perivascular and interstitial fibrosis and significant hypertrophy of myocytes indicated by diameters up to 31 m . a hcmtypical disarray was not detected . similar , no signs of cardiac storage diseases were found using different staining techniques . however , immunohistochemical staining showed an extensive active inflammation response of the myocardium ( figure 2 ) : highly increased 2leukocyteintegrins / infiltrates ( lfa1/cd11a+ and mac1/cd11b+ ) and mixed cellular infiltrates of both lymphocytes and macrophages ( cd45ropositive and hlapositive cells ) ( table 1).2 molecular biological analyses by nested polymerase chain reaction excluded the presence of known cardiotropic viruses including enterovirus , adenovirus , epsteinbarrvirus , human herpesvirus 6 , and parvovirus b19.3 magnetic resonance images of the heart and histological / immunohistochemical staining for fibrosis and inflammation . representative pictures showing a ) four chamber view and c ) shortaxis view of t1weighted magnetic resonance imaging scan . b ) four chamber view of a pronounced positive late gadolinium enhancementmagnetic resonance imaging scan . left corner : representative azanmallory staining image for fibrosis ( original magnification 100 ) , right corner : representative lfa1/cd11a+staining image for cellular infiltration ( original magnification 200 ) from the septum of the right ventricle and d ) short axis view of b. immunohistological staining of inflammatory cells of right ventricular septal biopsies . representative images depicting a ) mac1/cd11b+=infiltrates ( original magnification 200 ) ; b ) lfa1/cd11a+=infiltrates ( original magnification 200 ) ; c ) cd45ro cellular infiltrates ( original magnification 200 ) ; and d ) hla cellular infiltrates ( original magnification 100 ) present in the endomyocardial biopsy . quantification of active inflammation in endomyocardial biopsies based on the emb results , which evidenced a virusnegative active myocarditis , which was most probably the cause of the impairment of cardiac function , we initiated an immunosuppressive therapy with corticosteroid ( prednisolone 1 mg / kg / day ) and azathioprine ( 100 mg / day ) under blood count control and liver / kidney function control . after 4 weeks , the dose of prednisolone was tapered off every 4 weeks by 10 mg until a maintenance dose of 10 mg was reached.2 within the time frame of 3 months after starting the immunosuppressive treatment , the lvef continuously improved up to 52% , and the lvedd decreased to 56 mm . also , ntprobnp levels dropped to 968 pg / ml accompanied by regular levels of transaminases , crp , and leukocytes . in addition , her clinical symptoms improved . in summary , we show that external stress triggered by myocarditis can induce a transition from a hcmphenotype to a dilated cm ( dcm)phenotype . hypertrophic cardiomyopathy generally underlies a genetic disorder : around 4060% of all causes of hcm account for gene mutations of cardiac sarcomere proteins ( e.g. myosinbinding protein c , myosin heavy chain , cardiac troponin i and t , myosin light chain 3 and tropomyosin 1 chain ) . especially in infants and adolescents with isolated cardiac hypertrophy , more than 50% of all cases are due to genetic disorders.4 in addition , mixed genetic disorders causing hcm and rhythm diseases are also known and include the socalled prkag2 cardiac syndrome defined by the presence of hcm , ventricular preexcitation and tachyarrhythmia ( wpwsyndrome ) , and progressive conduction system disease.5 this genetic disorder , first described in a frenchcanadian family by gollob6 , underlies a mutation in the gene ( prkag2 ) encoding for the 2subunit of the heterotrimer ampactivating protein kinase.7 , 8 , 9 , 10 however , the presence of a prkag2 syndrome in our patient was already excluded years ago . in addition , the evaluation of other wellknown mutations able to cause hcm , including the screening for mutations of myh7 , mybpc3 , tnnt2 , tpm1 , tnni3 , myl3 , myl2 , csrp3 , pln , actc , and tnnc1 did not lead to a definite result in our patient . it is well accepted that in up to 30% of hcm , specific mutations can not be detected or are still unknown . in addition , up to 20% of patients with the phenotype of a significant lv hypertrophy do not belong to the classical form of hcm and comprise other genetic and nongenetic causes including storage diseases.11 , 12 forms of hcm can develop a dcmphenotype under stress conditions , but this is not very common and mostly seen in severe forms of hcm with lv obstructions . additional external stress includes emotional stress , toxic substances like alcohol or chemotherapeutics , pregnancy , ischemia , tachycardiainduced heart rhythm disturbances , and inflammation . matsumori et al . reported that viral myocardial infection can belong to these risk factors , too.13 the myocardial virus itself as well as inflammatory responses can lead to a deterioration of the hcm leading together with an increased intraventricular pressure to a lv dilatation.14 this mechanism has been described in patients with hcm and myocarditis most likely belonging to the worsening of cardiac function and to the induction of electrical imbalances.15 based on the emb findings , we concluded that our patient suffered from a virusnegative active myocarditis . thus , immunohistochemical analyses evidenced a significant , partly confluent lymphocyte infiltration , and increased expression of adhesion molecules . it also showed a marked infiltrate of macrophages and other inflammatory cells , but no increased specific cytotoxic t lymphocytes ( perforin positive ) were found . in opposite to the embs , the cardiac mri did not show specific signs for cardiac inflammatory processes . the mri resulted in the detection of fibrosis ( lge ) , which could belong to the hcm phenotype or to an additional inflammatoryinduced damage . fibrosis could be detected in embs taken from the lgeareas of the septum , too ( figure 1 b ) . however , only the further staining for inflammatory cells of these embs led to the definite diagnosis of a severe virusnegative active myocarditis ( figure 2 ) . these results are in agreement with the findings of others showing that cardiac mri sensitivity ( 76% ) , specificity ( 54% ) , and accuracy ( 68% ) has important limitations for detection of inflammatory processes and might not be sufficient to diagnose myocarditis in all cases.16 a negative mriresult does not automatically exclude a myocarditis and need further evaluation by emb , as concluded by the esc working group on myocardial and pericardial diseases.17 the embbased diagnosis of a severe virusnegative active myocarditis led us immediately to start with an immunosuppressive treatment including prednisolone and azathioprine for 6 months as recommended by caforio et al.17 in accordance to the timic study,18 we evidenced an improvement in lvef and a reduction in lvedd already 3 months after immunosuppressive treatment , correlating with a clinical improvement in nyha classification ( from class iii to i ) . in conclusion , this case shows that myocardial inflammation can contribute to a transition of a hypertrophic to a dilated phenotype , which can be reversible after induction of a specific antiinflammatory therapy .

granulocyte macrophage - colony stimulating factor ( gm - csf ) is a hematopoietic growth factor , which stimulates the proliferation of granulocytes and macrophages from bone marrow precursor cells . in autoimmune and inflammatory diseases , th17 cells have been considered as strong inducers of tissue inflammation . however , recent evidence indicates that gm - csf has prominent proinflammatory functions and that this growth factor ( not il-17 ) is critical for the pathogenicity of cd4 + t cells . therefore , the mechanism of gm - csf - producing cd4 + t cell differentiation and the role of gm - csf in the development of autoimmune and inflammatory diseases are gaining increasing attention . this review summarizes the latest knowledge of gm - csf and its relationship with autoimmune and inflammatory diseases . the potential therapies targeting gm - csf as well as their possible side effects have also been addressed in this review . abstractwe report the case of a 17yearold female patient with known hypertrophic cardiomyopathy and a wolffparkinsonwhite syndrome . she came to our department for further evaluation of a new diagnosed dilated cardiomyopathy characterized by an enlargement of the left ventricle and a fall in ejection fraction . clinically , she complained about atypical chest pain , arrhythmic episodes with presyncopal events , and dyspnea ( nyha iii ) during the last 6 months . noninvasive and invasive examinations including magnetic resonance imaging , electrophysiological examinations , and angiography did not lead to a conclusive diagnosis . therefore , endomyocardial biopsies ( embs ) were taken to investigate whether a specific myocardial disease caused the impairment of the left ventricular function . emb analysis resulted in the diagnosis of a virusnegative , active myocarditis . based on this diagnosis , an immunosuppressive treatment with prednisolone and azathioprine was started , which led to an improvement of cardiac function and symptoms within 3 months after initiating therapy . in conclusion , we show that external stress triggered by myocarditis can induce a reversible transition from a hypertrophic cardiomyopathy to a dilated cardiomyopathy phenotype . this case strongly underlines the need for a thorough and invasive examination of heart failure of unknown causes , including emb investigations as recommend by the actual esc position statement .

bacteriocins are toxins produced by the bacteria to inhibit the growth of similar or closely related bacterial strain(s ) during stress conditions . they are structurally , functionally , and ecologically diverse , produced by almost all major lineages of eubacteria and archaebacteria . ribosomal encoded bacteriocins are generally secreted in the extracellular milieu by the producers where they recognize specific receptors on the surface of susceptible or target cells . they induce toxicity in the target cells by different mechanisms like enzymatic nuclease ( dnase or rnase ) or pore formation in cytoplasmic membrane . their structure comprises of three distinct domain organizations : ( i ) a domain involved in recognition of specific receptor r , ( ii ) a domain involved in translocation t , and ( iii ) a domain responsible for their toxic activity c. molecular mass of ribosomal encoded bacteriocins vary from ~25 to 80 kda and are broadly classified into two groups , group a and b , based on their cross - resistance . these proteins have received increasing attention due to their potential use as preservatives in the food industry or in the therapeutic applications for clinical usage . xenorhabdus nematophila is a motile gram - negative bacterium belonging to the family enterobacteriaceae , and forms a symbiotic association in the gut of soil nematode from the family steinernematidae [ 6 , 7 ] . free - living forms of the bacterium have not yet been isolated from soil or water sources , which suggests that the symbiotic association may be essential for the survival of the bacteria in the environment . juvenile ( ijs ) enters digestive tract of the insect larva and subsequently penetrates into hemocoel of the host insect . the nematode can also gain access to the hemocoel via the respiratory spiracles or by penetrating directly through insect cuticle . bacteria , in turn , are essential for effective killing of the insect host and are required by the nematode to complete its life cycle [ 8 , 9 ] . growth in vitro is probably supported by the rich nutrient supply of the laboratory growth media and lack of competition that normally exists in the soil environment . as the bacteria enter stationary phase of growth cycle , they secrete several extracellular products , which include lipase(s ) , phospholipase(s ) , and protease(s ) , and several broad spectrum antibiotics that can be assayed in the culture media [ 10 , 11 ] . these extracellular products are believed to be secreted in the insect hemolymph when the bacteria enter stationary phase . these degradative enzymes break the macromolecules from insect cadaver to provide the developing nematode with nutrient supply , while the antibiotics suppress contamination of the cadaver by other microorganisms . cytoplasmic inclusion bodies , composed of highly expressed crystalline proteins , are also produced by the bacterium during stationary - phase growth . in our earlier study recombinant xenocin - immunity protein complex is toxic to six bacterial genus like bacillus , enterobacter , enterococcus , citrobacter , serratia , and stenotrophomonas . xenocin - immunity protein complex has atypical features which include the following . ( 1 ) tol box which has been replaced by ton box from n terminal end of translocation domain of xenocin . ( 2 ) there is only 30% similarity of xenocin with other bacteriocins . ( 3 ) size of its cognate immunity protein is 42 kda , whereas 1016 kda have been reported in other prokaryotic systems . immunity protein of x. nematophila is a fusion of two different domains , immunity domain and hemolysin domain . ( 4 ) immunity protein has atpase activity , although walker motif is missing in its primary amino acid sequence . ( 5 ) xenocin - immunity protein complex is secretory in nature without any signal sequence . in this study we have cloned , expressed , and purified all the possible domains of xcina and ximb genes . exogenous toxicity assays were performed with purified recombinant xenocin - immunity domain protein complex and other domains . in silico study of the immunity protein showed its similarity with hemolysin and purine ntpase like protein ; therefore , hemolysis and atpase assays were performed . finally , secondary structural analysis of recombinant xenocin - immunity domain protein complex , catalytic - immunity domain protein complex , immunity protein , and its hemolysin domains were performed with circular dichroism . all the chemicals were purchased from sigma ( sigma - aldrich ) except where otherwise mentioned . vector pqe30 , ni - nta agarose resin and qia quick spin columns were from qiagen ( germany ) . e. coli strains dh5 ( bethesda research laboratories ) were used as the host for cloning . e. coli bl 21(de3 ) plyss strain from novagen and m 15 strain from qiagen were used in the expression studies . the plasmid vector pgem - t easy from promega ( madison , usa ) were used for pcr cloning . ampicillin , kanamycin , and chloramphenicol were used in the concentration of 100 , 35 , and 25 g ml , respectively . phylogenetic analysis of immunity protein was done by a method as described earlier . briefly , amino acids of all the protein sequences that matched with immunity protein were aligned by clustalw ( mac vector 7.0 ) , and a tree was constructed using neighbour - joining method , with the best tree mode in the mac vector version 7.0 ( oxford molecular , england ) program . all the constructs were amplified from the 4.3 kb genomic dna fragment of x. nematophila . the orf 1 encoding xcina gene and partial orf 2 encoding immunity domain of ximb gene were obtained by pcr amplification using primer 1 with a bamhi site at the 5 end and a reverse primer 6 with hindiii site at the 3 end . the amplified product ( 2 kb ) was ligated in pgem - t easy and pqe30 vector , producing pjc5 and pjc6 plasmids , respectively . the catalytic domain ( 318 bp ) of xcina gene was cloned with the immunity domain ( first 270 bp of ximb gene ) of ximb gene using primer pair 2 and 6 as described earlier . primer 1 with a bamhi site at the 5 end and a reverse primer transb with ncoi site at the 3 end were used to amplify the translocation domain of xcina gene . amplified product of 1 kb was ligated in pgem - t easy vector and pqe30 vector , producing pjc7 and pjc8 plasmids , respectively . primer 1 with a bamhi site at the 5 end and a reverse primer recb with hindiii site at the 3 end were used to amplify the translocation - receptor domain of xcina gene . the amplified product ( 1.5 kb ) was ligated in pgem - t easy vector and pqe30 vector producing pjc9 and pjc10 plasmids , respectively . primer 3 with bamhi site at the 5 end and a reverse primer 5 with hindiii site at the 3 end used for ximb cloning and forward primer 4 without any restriction site and backward primer 7 without any restriction site were used for cloning ximb hemolysin domain . the amplified products of 1 kb and 700 bp were ligated in pgem - t easy vector producing pjc11 and pjc12 plasmids . the 1 kb amplified product was further ligated in pqe30 and 700 bp amplified product was ligated in pqe31 expression vector producing pjc13 and pjc14 plasmids , respectively . a 2.330 kb dna fragment containing both xcina and immunity domain of ximb gene with native promoters was amplified using xenocinf1 ( 300 bp upstream of start codon of xcina locus ) and primer 6 and cloned in pgem - t easy vector producing pxim construct . the empty pgem - t easy vector designated as pgem was used as control . the plasmids pjc6 , pjc8 , pjc10 , pjc13 , and pjc14 were transformed in m15 cells where as pjc4 was transformed in e. coli bl 21(de3 ) plyss cells . the resulting strains jc4 , jc6 , jc8 , jc10 , jc13 , and jc14 were used for expression and purification of recombinant proteins under the control of iptg inducible t7 promoter as per the protocol described earlier . briefly , overnight grown cultures were diluted 100 fold in fresh 50 ml lb medium and grown till the od600 reached 0.5 . culture was induced by adding 1 mm final concentration of iptg and incubated at 30c for 6 hours . cells were harvested and washed with 40 ml of cold and 50 mm sodium phosphate buffer , ph 8 , containing 300 mm nacl and 50 mm benzamidine ( buffer a ) . the cell pellet was suspended in 25 ml of buffer a and cells were disrupted by sonication at 4c . the cell lysate was centrifuged at 12000 g for 30 min at 4c in a rc5 plus centrifuge , and the 6xhis - tagged recombinant proteins or protein complexes in the soluble fractions were purified as follows . the supernatant from the previous step was loaded on ni - nta agarose column preequilibrated with buffer a at 4c . the column was washed extensively with buffer a , containing 2550 mm imidazole , and the protein / protein complex was eluted with buffer a containing 300 mm imidazole . fractions containing pure protein or protein complex were concentrated using centricon ( millipore pm10 ) . recombinant protein or protein complexes were dialyzed overnight against 100 volumes of 50 mm sodium phosphate buffer , ph 8 , and the final preparations were stored at 20c in the presence of 15% glycerol . to study the neutralizing effect of the immunity domain protein , xcina gene was cloned with its native promoter along with immunity domain of ximb gene which gave rise to pxim . empty pgem - t easy vector was considered as control and transformed in e. coli dh5 to give rise to gem strain . overnight grown strains gem and xim were subcultured in fresh medium and incubated till the od600 reached 0.5 . the cultures were diluted in fresh medium 1 : 100 and induced with 0.3 g ml of mitomycin c ( an inducer of xenocin native promoter ) . the optical densities of the cultures were monitored at 600 nm during different intervals . the bacteriostatic activity of purified recombinant proteins / complexes were determined by the protocol as described earlier . briefly , lb agar plates without antibiotics were overlaid with 3 ml of soft nutrient agar containing indicator e. coli dh5 strain grown in m9 medium , and the protein complex was applied to sterile disks . the plates were incubated overnight at 37c , and the sizes of clearance zones were recorded . freshly isolated rabbit blood cells were washed thrice with phosphate buffer saline ( pbs ) by centrifuging at 1000 g , 4c for 10 minutes . washed erythrocytes were resuspended in pbs to make a final concentration of 4% . the same volume ( 100 l ) of protein ( 5 m ) sample dissolved in pbs and erythrocytes suspension were added into wells of 96-well plate . the plate was then incubated at 37c for 1 hr and centrifuged at 1000 g for 5 minutes . the resulting supernatant was transferred to new wells , and the absorbance was determined at 540 nm on a continuous spectrum microtitre plate reader . protein samples of different concentrations were incubated with 0.2 ci of [ -p ] labelled atp ( 6000 ci/ mmol , perkinelmer life sciences , usa ) in a buffer containing 20 mm tris - hcl ( ph 8.0 ) , 1 mm mgcl2 , 100 mm kcl , 8 mm dtt , and 80 g / ml of bsa in a total reaction volume of 10 l . samples were incubated at 37c for 30 minutes . at the end of the reaction , 1 l of the reaction mixture was spotted on a polyethyleneimine thin - layer chromatography ( tlc ) plate ( sigma - aldrich , usa ) and air - dried . chromatography was performed using 0.5 m licl and 1 m hcooh as the running solvent . the far - uv cd spectrum was recorded between 190 and 260 nm ( 500 l sample volume ) on a jasco j-810 spectropolarimeter equipped with a jasco peltier temperature controller at 25c using 1 mm optical path length quartz cells and the step size was 0.5 nm with 1 nm bandwidth at a scan speed of 20 nm minute . averages of 5 scans were obtained for blank and protein spectra , and the data was corrected for buffer contribution . the results were expressed as mean residue ellipticity in units of degree / cm / dmol . phylogenetic analysis of the xenocin and its immunity protein was done by preparing phylogenetic tree , using neighbour - joining method . results showed that immunity protein formed a separate cluster in the very beginning as shown figure 1(b ) . further , xcina gene was cloned along with n terminal immunity domain ( 10 kda ) of ximb gene . purification of recombinant xenocin - immunity domain ( 10 kda ) protein complex from jc6 strain was done by ni - nta chromatography under native conditions . one was at the position corresponding to 66 kda and another below 14 kda protein marker as shown in figure 2(a ) which corroborates with the size of xenocin and immunity domain of immunity protein , respectively . purification of recombinant catalytic - immunity domain protein complex with ni - nta chromatography under native conditions from jc4 strain also showed two bands corresponding to the size of catalytic domain of xcina and immunity domain of ximb as shown in figure 2(b ) . purification of recombinant translocation domain of xcina gene from jc8 strain with ni - nta chromatography showed multiple bands in sds - page as shown in figure 2(e ) . further , western blot was performed using purified fraction , probed with anti - his antibody , and the result showed single band at 38 kda which corresponds to the size of recombinant translocation domain protein as shown in figure 2(f ) . purification of recombinant translocation - receptor domain protein with ni - nta chromatography from jc10 strain showed two prominent bands in sds - page . upper band corresponded to ~52 kda whereas lower band corresponded to ~28 kda as shown in figure 2(g ) . western blot was performed using whole cell lysate and the purified fraction , probed with anti - his antibody , which showed a single band at 52 kda corresponding to a size of recombinant translocation - receptor domain protein as shown in figure 2(h ) . purification of recombinant full length immunity protein , as well as its hemolysin domain protein from jc13 and jc14 strains , showed less but stable expression as shown in figures 2(c ) and 2(d ) , respectively . neutralization of endogenous toxicity of xcina by immunity domain of ximb gene was determined by endogenous assay . endogenous assay with xim strain ( harboring pxim containing xcina with its native promoter and first 85 amino acid residues of ximb gene ) and gem strain ( harboring empty pgem t - easy vector ) in the presence of mitomycin c showed the same growth profile as shown in figure 3(a ) . exogenous toxicity assay was performed with purified recombinant xenocin - immunity domain ( 10 kda ) complex using e. coli dh5 as target cells . the zone of inhibition was observed as shown in figure 3(b ) ( i ) . purified catalytic - immunity domain protein complex was used to study for the bacteriostatic effect in the exogenous assays . the zone of inhibition was not observed in this case as shown in figure 3(b ) ( ii ) . similar results were observed when full length immunity protein ( 42 kda ) encoded by ximb gene or its hemolysin domain ( 32 kda ) was used for exogenous assay as shown in figure 3(b ) ( iii ) and ( iv ) , respectively . moreover , as expected zone of inhibition was not observed in buffer control experiment as shown in figure 3(b ) ( v ) . protein - protein blast results ( http://www.ncbi.nlm.nih.gov/blast/ ) of the immunity protein showed its similarity with hemolysin ( aaf42109 ) and purine ntpase like protein ( data not shown ) . hemolytic assay with fresh rabbit red blood cells was performed with purified full length immunity protein as well as its immunity and hemolysin domain . results showed that none of the protein had hemolytic activity ( data not shown ) . atpase assay was performed with purified recombinant full length immunity protein , its immunity domain , and hemolysin domain . atpase activity was not detected in recombinant immunity domain , hemolysin domain protein , and even in the purified bsa which was used as negative control as shown in figure 4(a ) lane 1 . however , full length recombinant immunity protein ( 42 kda ) showed atpase activity with increasing concentration of protein and was comparable to the atpase activity of purified groel protein of x. nematophila which was used as the positive control , as shown in figure 4(a ) lane 2 . the far uv spectra of purified recombinant xenocin - immunity domain ( 10 kda ) protein complex , catalytic - immunity domain protein complex , immunity , and its hemolysin domain were recorded at 25c as shown in figure 5 . recombinant xenocin - immunity domain protein complex was found to contain 41% -helical structure and 21% -sheet . catalytic - immunity domain protein complex was found to contain 51% -sheet and only 7% -helical structure . in case of full length immunity protein 30% of the secondary structure hemolysin domain of the immunity protein also had the same secondary conformation with 30% -helical and 11% -sheet content . recombinant xenocin-(66 kda- ) immunity ( 42 kda ) protein complex has a broad range bacteriostatic property , inhibiting the growth of six insect gut resident bacterial species . due to only 30 - 31% primary sequence similarity with other bacteriocins , xenocin from the x. nematophila forms a distinct cluster in phylogenetic tree . phylogenetic analysis of immunity protein showed similar results , in which immunity protein from x. nematophila forms a separate cluster in the very beginning . this could be due to variable length of immunity protein from x. nematophila . cognate immunity protein of xenocin consists of 368 amino acid residues and is a unique fusion of two different domains . its n terminal ( first 85 amino acid residues ) showed similarity with immunity protein from other prokaryotic systems , whereas the c terminal showed similarity with hemolysin ( n. meningitidis accession no . three - dimensional structure of xenocin has been recently deciphered by homology modelling in my lab . it is a multidomain protein which consists of 576 amino acid residues . from its n terminal 1327 amino acid residues form translocation domain ( t ) , 328476 amino acid residues form the middle receptor domain ( r ) , and amino acid residues from 477576 form the catalytic domain ( c ) at the c terminal . while cloning xcina gene alone in expression vector , not a single transformant was observed . one reason for this result could be the leaky expression of toxic xcina gene . to address this question , xcina gene was cloned along with n terminal immunity domain ( 10 kda ) of ximb gene . when the recombinant protein from jc6 strain was purified by ni - nta chromatography , xenocin was visible in sds - page at position corresponding to 66 kda whereas immunity domain of immunity protein was observed below 14 kda protein marker . this result showed that n terminal immunity domain ( 10 kda ) of immunity protein ( 42 kda ) encoded by ximb gene is enough to bind with and neutralize the in vivo toxic effect of xcina gene . to confirm this result and to map the minimum functional domain of immunity protein required to abolish the xcina gene toxicity in vivo , first 85 amino acids of immunity protein were cloned along with xcina gene under its native promoter . same growth profile of xim strain ( harboring pxim ) and gem strain ( harboring empty pgem t - easy vector ) in the presence of mitomycin c confirmed that first 85 amino acid residues of ximb gene were able to neutralize the toxic activity of xenocin in vivo . further , purification of recombinant catalytic - immunity domain protein complex with ni - nta chromatography under native conditions from jc4 strain confirmed the minimal domains of xcina and ximb genes that could be expressed and purified . as we were unable to express xcina gene alone which is composed of translocation , receptor , and catalytic domain an attempt had been made to clone , express , and purify the translocation domain alone or along with receptor domain of xcina gene . in native conformation , translocation domain of bacteriocin like colicin e3 interacts with catalytic domain of e3 and immunity protein via receptor domain , and this interaction further provides stability to the translocation domain . however , in recombinant translocation domain of xcina , receptor and catalytic domains as well as immunity protein were missing which may be probably made it to attain an open conformation and be susceptible to proteases from the host cells . hence , during the purification of translocation domain from pjc8 , multiple bands were observed in sds - page . however , western blot with purified fraction when probed with anti - his antibody showed a single band corresponding to the size of translocation domain which confirmed the expression of translocation domain alone . further , purification of recombinant translocation - receptor domain protein with ni - nta chromatography from jc10 strain , showed two prominent bands in sds - page rather than multiple bands . upper band corresponded to ~52 kda whereas lower band corresponded to ~28 kda . western blot using whole cells and purified fraction when probed with anti - his antibody showed a single band at 52 kda which is corresponding to a size of recombinant translocation - receptor domain protein . therefore , we inferred that recombinant translocation domain along with receptor domain of the xcina gene was more stable as compared to the recombinant translocation domain alone , but due to the absence of catalytic domain and immunity protein it was still prone to proteases of the host cell . endogenous toxicity assays with xim and gem strains were performed to identify the minimum domain required to neutralize the xcina toxicity . results showed that first 85 amino acids of the immunity protein were able to neutralize xenocin endogenous toxicity as both strains had the same growth profile . moreover , from this experiment we inferred that expression of cognate immunity protein domain and its binding to the catalytic domain of xenocin occurs in the host cell simultaneously . receptor domain in bacteriocin plays a major role in the exogenous toxic effect , as it is the first domain which binds to ligand present on the surface of its target cells . binding is followed by import of bacteriocin into the cells with the assistance of either ton proteins ( exbb , exbd , and tonb ) or tol proteins ( tola , -b , -q , and -r ) of periplasm and facilitate the translocation of the catalytic domain of bacteriocins into the periplasmic space of the target cell for further processing [ 5 , 1618 ] . interaction of bacteriocins either with ton or tol proteins of periplasm occurs via ton or tol box present at their n terminal end of the translocation domain . interestingly , although xenocin has rnase activity , tol box in its translocation domain has been replaced by ton box . this could be due to the domain swapping which generally occurs during the horizontal gene transfer in prokaryotes . in the colicin e3-immunity complex , binding of the receptor domain of e3 to the surface protein on target cells leads to the conformational changes which assisted the immunity protein to dissociate from the protein complex [ 4 , 18 ] . the immunity protein from x. nematophila is a unique fusion of immunity domain ( 10 kda ) and hemolysin domain ( 32 kda ) protein , and endogenous assay confirmed that immunity domain of ximb gene is enough to neutralize the detrimental effect of xcina gene . therefore , exogenous assay was performed with purified recombinant xenocin - immunity domain ( 10 kda ) complex using e. coli dh5 as target cells to determine translocational ability of xenocin - immunity domain ( 10 kda ) complex into the cytoplasm of target cells . clear zone of inhibition was observed in the exogenous toxicity assay due to the detrimental effect of xenocin - immunity domain ( 10 kda ) protein complex . this result confirmed the functionality of translocation as well as receptor domain of xenocin and their individual roles in internalization of xenocin into the cytoplasm of target cells . moreover , it also confirmed that hemolysin domain ( 32 kda ) of ximb gene has no role in the internalization of xenocin as well as in exogenous toxicity . catalytic domain of xcina gene along with immunity domain of ximb gene was the minimum domain which could be expressed and purified . therefore , purified catalytic - immunity domain protein complex was studied for the bacteriostatic effect in the exogenous assays . results showed that zone of inhibition was not observed , which again confirmed the role of translocation as well as receptor domain of xcina gene for its internalization into the cytoplasm on the target cells . similar results were observed when full length immunity protein ( 42 kda ) encoded by ximb gene or its hemolysin domain ( 32 kda ) was used for exogenous assay . moreover , as expected zone of inhibition was not observed in the experiment in which buffer was used as a negative control . to neutralize the detrimental effect of bacteriocins , the molecular weight of the immunity protein in other prokaryotic systems is between 10 and 16 kda ; however , immunity protein encoded by ximb gene corresponds to 42 kda . protein - protein blast using blastp ( http://www.ncbi.nlm.nih.gov/blast/ ) of the immunity protein showed similarity with hemolysin ( aaf42109 ) and purine ntpase like protein . hemolytic assay with fresh rabbit red blood cells was performed with purified full length immunity protein as well as its immunity and hemolysin domains . due to the partial primary amino acid sequence similarity with hemolysin protein , none of the protein / protein domains showed hemolytic activity . as immunity protein also showed similarity with protein like purine ntpase 's atpase assay was performed with purified recombinant full length immunity protein , its immunity domain , and hemolysin domain . although full length immunity protein showed atpase activity , in silico analysis did not show walker motif in its primary amino acid sequence , which is generally present in the proteins having atpase activity . possibility of atpase activity in immunity protein towards the secretion of recombinant xenocin - immunity protein complex can not be ruled out at this stage as none of the proteins have any signal sequence at their either ends . secondary structure analysis of purified recombinant xenocin - immunity domain ( 10 kda ) protein complex , catalytic - immunity domain protein complex , immunity , and its hemolysin domain was done with far uv spectra . high percentage of -helical structure of xenocin - immunity complex is attributed due to the helix turn helix structure of receptor domain of xenocin which corroborates with its recently deciphered three - dimensional structure . catalytic - immunity domain protein complex was found to contain 51% -sheet and only 7% -helical structure . this open conformation with high -sheet might be beneficial to bind with its substrate ( rna ) and act upon it [ 20 , 21 ] . in case of full length immunity protein 30% of hemolysin domain of the immunity protein also had the same secondary conformation with 30% -helical and 11% -sheet content . since immunity protein is a novel fusion of two different domains the role of its secondary is not deciphered yet . comet lysis buffer ( 2.5 m sodium chloride , 100 mm edta ( ph 8.0 ) , 1% sarkosyl , 10 mm tris hcl ( ph 8.0 ) , 10% dmso , 10% triton x-100 ) . comet alkali solution ( 0.3 m sodium hydroxide , 1 mm edta ) . cryopreservation medium ( 50% foetal calf serum [ fcs ] , 10% dmso , 40% dulbecco 's modified eagle 's medium [ dmem ] ) . culture medium for hepg2 cells ( dmem , 10% fcs , 50 u / ml penicillin , 50 u / ml streptomycin , 2 mm l - glutamine ) . all chemicals were purchased from sigma chemicals co. , dorset , uk . hepg2 cells were obtained from the european cell culture collection ( eccc , uk ) . hepg2 cells are human caucasian hepatocyte carcinoma cells taken from a primary hepatoblastoma ( liver biopsy ) from an 11-year - old male from argentina in 1979 . the frozen vials of the cells were held at room temperature for 1 min and then placed in a 37c incubator for 1 min until thawed and the cells were pipetted into a flask containing 10 ml of pre - warmed dmem . hepg2 cells were grown in dmem supplemented with 10% fcs , 2 mm glutamine , 100 g / ml penicillin and 100 g / ml streptomycin . the cells were fed three times a week and split when confluence was reached . to split the cells , the medium was removed , the cells were washed with hank 's buffered salt solution ( hbss ) , and then gently harvested with 1 ml detachment trypsin neutralising solution . once all the cells were loosened from the plate , 1 ml of detachment trypsin neutralising solution was added . the cell suspension was pipetted into a sterile falcon tube and centrifuged at 1,200 rpm for 5 min . the supernatant was removed and the pellet was gently resuspended in an appropriate volume of medium . cells were plated in a multi - well plate : 1 ml at 510 cells / ml in dmem . supplemented with 10% fcs , 2 mm glutamine , 100 g / ml penicillin and 100 g / ml streptomycin . the cells were allowed to attach for 24 h at 37c . then the medium was replaced by demem containing 25 m or 50 m h2o2 and duplicate cultures were incubated at 37c for 5 min , 30 min , 40 min , 1 h , or 24 h. at the end of incubation the cells were harvested and used in comet assay analysis of dna damage . the principle of the comet assay is that smaller dna molecules migrate faster in an electric field than larger molecules . the treated cells are encapsulated in gel and lysed by alkali , which also denatures the dna . subsequent electrophoresis causes migration of the dna . while the undamaged dna appears as a head , fragmented dna move faster , giving the characteristic appearance of a comet tail ( fig . 1 ) . levels of dna damage after exposure to hydrogen peroxide evaluated by the comet assay . fig . partly frosted microscope slides were pre - coated with a smear of 1% low melting - point agarose ( lmpa ) and allowed to dry for 1 h at 37c . the slides were then placed on a bed of ice to pre - cool them before adding an agarose sandwich . an aliquot of 170 l of 1% lmpa was placed onto the slide and immediately covered with a coverslip . whilst this was setting , an aliquot of 170 l of the agarose / lymphocyte mix was placed on top of the first layer and again covered immediately with a coverslip . once set , the coverslip was removed and a final layer of 170 l 1% lmpa was applied to the existing gel to form an agarose sandwich , with the cells in the middle layer . the lights were switched off and the remainder of the assay was performed using indirect light . flowchart showing the comet assay for single - cell gel electrophoresis to determine dna damage ( 12 ) . a pyrex tray containing 500 ml of ice - cold lysis buffer was placed onto a bed of ice and the slides were gently lowered into it . after 1 h , the slides were removed and washed by placing them in 500 ml ice - cold pbs ( again in a pyrex tray on a bed of ice ) and allowing them to sit for 15 min . this removed the salt from the lysis buffer and prepared the slides for the next step . a horizontal electrophoresis tank was surrounded by ice and filled with 2 l of ice - cold lysis buffer . then the slides were gently lowered into a horizontal electrophoresis tank filled with 2 l of cold lysis buffer and surrounded by ice . the slides were placed for 30 min to allow the dna to unwind in the alkali buffer . next , the slides were removed and rinsed by placing them in 500 ml of ice - cold neutralising buffer for 10 min and then in 500 ml of ice - cold pbs for 15 min . dna damage was measured using the comet assay and expressed as olive tail moment ( otm ) . results are given as meansem for the 50 cells ( 25 per slide ) . to determine cell viability ( 14 ) , 100 l of cell suspension was mixed 1:1 with trypan blue stain ( 0.4% w / v , biowhittaker ) and pipetted into a neubauer haemocytometer ( vwr scientifics , west chester , pa ) . if membrane integrity has been compromised ( dead cells ) , the cells absorb the dye and appear blue . in our study , if cell viability < 80% , the cells were discarded and a new batch was started . for the experiments we report , one - way anova was performed when more than two groups were compared with a single control . differences between individual groups were assessed by a dunnett post hock test , using prism software ( version 4 ) . the slide was visualised using either a biorad mrc 600 confocal microscope or a leica tcs sp2 uv confocal microscope . images were obtained with the biorad using a krypton / argon laser , 20 magnification , and excitation wavelength at 568 nm , lens aperture of 0.4 and z series slices of 3-m steps . images were obtained with the leica using a 543 helium / neon ( he / ne ) laser at 55% power and 20 magnification , and the emission was collected using a detection window of 570655 nm wavelength . comet lysis buffer ( 2.5 m sodium chloride , 100 mm edta ( ph 8.0 ) , 1% sarkosyl , 10 mm tris hcl ( ph 8.0 ) , 10% dmso , 10% triton x-100 ) . comet alkali solution ( 0.3 m sodium hydroxide , 1 mm edta ) . cryopreservation medium ( 50% foetal calf serum [ fcs ] , 10% dmso , 40% dulbecco 's modified eagle 's medium [ dmem ] ) . culture medium for hepg2 cells ( dmem , 10% fcs , 50 u / ml penicillin , 50 u / ml streptomycin , 2 mm l - glutamine ) . all chemicals were purchased from sigma chemicals co. , dorset , uk . hepg2 cells were obtained from the european cell culture collection ( eccc , uk ) . hepg2 cells are human caucasian hepatocyte carcinoma cells taken from a primary hepatoblastoma ( liver biopsy ) from an 11-year - old male from argentina in 1979 . the frozen vials of the cells were held at room temperature for 1 min and then placed in a 37c incubator for 1 min until thawed and the cells were pipetted into a flask containing 10 ml of pre - warmed dmem . hepg2 cells were grown in dmem supplemented with 10% fcs , 2 mm glutamine , 100 g / ml penicillin and 100 g / ml streptomycin . the cells were fed three times a week and split when confluence was reached . to split the cells , the medium was removed , the cells were washed with hank 's buffered salt solution ( hbss ) , and then gently harvested with 1 ml detachment trypsin neutralising solution . once all the cells were loosened from the plate , 1 ml of detachment trypsin neutralising solution was added . the cell suspension was pipetted into a sterile falcon tube and centrifuged at 1,200 rpm for 5 min . the supernatant was removed and the pellet was gently resuspended in an appropriate volume of medium . cells were plated in a multi - well plate : 1 ml at 510 cells / ml in dmem . supplemented with 10% fcs , 2 mm glutamine , 100 g / ml penicillin and 100 g / ml streptomycin . the cells were allowed to attach for 24 h at 37c . then the medium was replaced by demem containing 25 m or 50 m h2o2 and duplicate cultures were incubated at 37c for 5 min , 30 min , 40 min , 1 h , or 24 h. at the end of incubation the cells were harvested and used in comet assay analysis of dna damage . the principle of the comet assay is that smaller dna molecules migrate faster in an electric field than larger molecules . the treated cells are encapsulated in gel and lysed by alkali , which also denatures the dna . subsequent electrophoresis causes migration of the dna . while the undamaged dna appears as a head , fragmented dna move faster , giving the characteristic appearance of a comet tail ( fig . 1 ) . levels of dna damage after exposure to hydrogen peroxide evaluated by the comet assay . fig . partly frosted microscope slides were pre - coated with a smear of 1% low melting - point agarose ( lmpa ) and allowed to dry for 1 h at 37c . the slides were then placed on a bed of ice to pre - cool them before adding an agarose sandwich . an aliquot of 170 l of 1% lmpa was placed onto the slide and immediately covered with a coverslip . whilst this was setting , an aliquot of 170 l of the agarose / lymphocyte mix was placed on top of the first layer and again covered immediately with a coverslip . once set , the coverslip was removed and a final layer of 170 l 1% lmpa was applied to the existing gel to form an agarose sandwich , with the cells in the middle layer . the lights were switched off and the remainder of the assay was performed using indirect light . flowchart showing the comet assay for single - cell gel electrophoresis to determine dna damage ( 12 ) . a pyrex tray containing 500 ml of ice - cold lysis buffer was placed onto a bed of ice and the slides were gently lowered into it . after 1 h , the slides were removed and washed by placing them in 500 ml ice - cold pbs ( again in a pyrex tray on a bed of ice ) and allowing them to sit for 15 min . this removed the salt from the lysis buffer and prepared the slides for the next step . a horizontal electrophoresis tank was surrounded by ice and filled with 2 l of ice - cold lysis buffer . then the slides were gently lowered into a horizontal electrophoresis tank filled with 2 l of cold lysis buffer and surrounded by ice . the slides were placed for 30 min to allow the dna to unwind in the alkali buffer . next , the slides were removed and rinsed by placing them in 500 ml of ice - cold neutralising buffer for 10 min and then in 500 ml of ice - cold pbs for 15 min . dna damage was measured using the comet assay and expressed as olive tail moment ( otm ) . to determine cell viability ( 14 ) , 100 l of cell suspension was mixed 1:1 with trypan blue stain ( 0.4% w / v , biowhittaker ) and pipetted into a neubauer haemocytometer ( vwr scientifics , west chester , pa ) . if membrane integrity has been compromised ( dead cells ) , the cells absorb the dye and appear blue . in our study , if cell viability < 80% , the cells were discarded and a new batch was started . for the experiments we report , cell viability was 9095% after culturing and before starting the comet assay . one - way anova was performed when more than two groups were compared with a single control . differences between individual groups were assessed by a dunnett post hock test , using prism software ( version 4 ) . the slide was visualised using either a biorad mrc 600 confocal microscope or a leica tcs sp2 uv confocal microscope . images were obtained with the biorad using a krypton / argon laser , 20 magnification , and excitation wavelength at 568 nm , lens aperture of 0.4 and z series slices of 3-m steps . images were obtained with the leica using a 543 helium / neon ( he / ne ) laser at 55% power and 20 magnification , and the emission was collected using a detection window of 570655 nm wavelength . cell viability following different treatments was 9095% and no floating cells were noticed in the medium . we analysed dna damage by the comet assay . following single - cell electrophoresis , the lengths of the comets ( dna trails ) depended on the treatment , with longer tails indicating more dna damage . however , cells incubated with h2o2 for 24 h appeared similar to untreated control cells ( fig . comet assay of the effect of duration of incubation with hydrogen peroxide on dna damage : untreated hepg2 cells ( a ) and hepg2 cells treated with 50 m h2o2 for 5 min ( b ) , 1 h ( c ) or 24 h ( d ) . the extent of dna damage , measured in otm , increased rapidly and significantly ( p<0.001 ) from baseline levels of 1.4 otm0.2 sem to 13.41.3 otm after 5 min of treatment with 25 m h2o2 and 15.50.6 otm after 5 min of treatment with 25 m h2o2 ( fig . the extent of damage increased further with time but at a slower rate , reaching about 2530 otm after 1 h of treatment . however , at the 24-h time - point the values for treatment with 25 and 50 m h2o2 ( 5.91.3 and 3.70.6 otm , respectively ) were not significantly higher than the baseline levels . the extent of dna damage was higher for cells treated with 50 m h2o2 than for those treated with a 25 m concentration at the 5 , 30 , 40 and 60 min time - points ( fig . hepg2 cells were treated with 25 m or 50 m h2o2 for 5 , 30 , 40 min , 1 h or 24 h and dna damage was assessed by the comet assay . the values are the meansem of 50 cells from two pooled wells ( 25 cells per slide ) . when formation of ros exceeds the biological defence capacity , the result is oxidative stress and tissue injury ( 15 ) . h2o2 can cause oxidative stress because it uses water channels ( aquaporins ) to rapidly cross - cell membranes ( 20 ) , reach the nucleus and cause damage to dna by generating hydroxyl - free radicals ( oh ) ( 21 ) . these radicals attack dna at the sugar residue of the dna backbone , leading to single strand breaks . they also modify purines and pyrimidines to their hydroxyl derivatives , such as 8-hydroxyguanine ( 16 ) . one study examined dna damage resulting from treatment of cultured human leukocytes with h2o2 concentrations of 25 , 50 , 100 and 200 m for 4 h. dna damage , assessed by the comet assay , increased significantly with increasing dose ( 17 ) . in our study , we treated hepg2 cells with 25 m or 50 m h2o2 for different durations : 5 min , 30 min , 40 min or 1 h. dna damage was both dose and duration dependent . dna damage increased with increasing time of incubation with h2o2 , but only between 5 and 60 mins of incubation . after 24 h of incubation with h2o2 , the extent of dna damage was not different from that in control cells , quite likely due to the action of dna repair mechanisms . as we did not use incubation times longer than an hour but shorter than 24 h , we do not know at what time point the effects of such repair mechanisms became evident . however , a similar study on colonocytes ( 18 ) showed that at a h2o2 concentration of 15 mm the dna damage was reduced when incubation times were > 30 min . similarly , treatment of human lymphocytes with 20 m h2o2 for 5 min induced dna damage but incubation of the cells for 2 h led to a considerable decrease in dna damage ( 19 ) . based on these reports we suggest that dna damage in our setup starts to decrease shortly after 1 h of incubation . in conclusion , the extent of dna damage was dose - dependent and incubation time - dependent . dna damage increased with time but later decreased , likely due to metabolism of h2o2 to water and the effects of dna repair . the authors have not received any funding or benefits from industry to conduct this study .

xenorhabdus nematophila , a gram - negative bacterium belonging to the family enterobacteriaceae is a natural symbiont of a soil nematode from the family steinernematidae . in this study cloning , expression , and purification of broad range iron regulated multidomain bacteriocin called xenocin from x. nematophila ( 66 kda , encoded by xcina gene ) and its multidomain immunity protein ( 42 kda , encoded by ximb gene ) have been done . xcina - ximb ( n terminal 270 bp ) , translocation , and translocation - receptor domain of xcina , ximb , and its hemolysin domain were cloned , expressed , and purified by single step ni - nta chromatography under native conditions . in the functional characterization , neutralization of xcina toxicity by immunity domain of ximb gene was determined by endogenous assay . exogenous toxic assays results showed that only the purified recombinant xenocin - immunity domain ( 10 kda ) protein complex had toxic activity . atypical cognate immunity protein ( 42 kda ) of xenocin was fusion of immunity domain ( 10 kda ) and hemolysin domain ( 32 kda ) . in silico analysis of immunity protein revealed its similarity with hemolysin and purine ntpase like proteins . hemolytic activity was not observed in immunity protein or in its various domains ; however , full - length immunity protein lacking walker motif showed atpase activity . finally , using circular dichroism performed secondary structural analyses of all the recombinant proteins / protein complexes . backgroundhydrogen peroxide ( h2o2 ) is a common reactive oxygen intermediate generated by various forms of oxidative stress.aimthe aim of this study was to investigate the dna damage capacity of h2o2 in hepg2 cells.methodscells were treated with h2o2 at concentrations of 25 m or 50 m for 5 min , 30 min , 40 min , 1 h , or 24 h in parallel . the extent of dna damage was assessed by the comet assay.resultscompared to the control , dna damage by 25 and 50 m h2o2 increased significantly with increasing incubation time up to 1 h , but it was not increased at 24 h.conclusionsour findings confirm that h2o2 is a typical dna damage - inducing agent and thus is a good model system to study the effects of oxidative stress . dna damage in hepg2 cells increased significantly with h2o2 concentration and time of incubation but later decreased likely due to dna repair mechanisms and antioxidant enzymes .

telemedicine utilization is rising due to the increased availability and decreased cost of communication technology , in parallel with growing recognition of key areas of health care that may benefit from its use . although phone communication continues to be a common form of patient - provider communication , internet - based video communication is being deployed in several settings as well . the portion of the population with internet access is rapidly growing74% of english - speaking americans have access , an absolute increase of 30% from 2000 to 2009 . given the widespread availability of internet access and the decreasing costs of video communication via computer , it is important to understand preferences and potential barriers , from the patient perspective , to facilitate telemedicine implementation . telemedicine can be employed in a synchronous or asynchronous manner . in asynchronous telemedicine , also known as store and forward , health information is obtained and communicated between visits , at which point discussion of that information may occur . this aspect of telemedicine is already commonly practiced in many settings ; examples include remote transmission of information from specialists ( such as radiology ) as well as patient use of email or weblogs for conveying information . by comparison , synchronous telemedicine involves real time interaction between provider and patient , such as by telephone or video . use of this method is growing in several settings as follows : providing acute decision making , such as in telestroke ; facilitating intermittent , low - level management of chronic illnesses ; increasing accessibility for patients who are either geographically isolated or find local travel challenging ; enabling patients with uncommon diseases to access specialists [ 4 , 5 ] . the potential benefits of synchronous telemedicine include improved access to healthcare , reduced waiting times for appointments , and increased patient adherence to chronic illness treatment plans . chronic diseases account for 75% of healthcare spending and represent a key target for both cost reduction and care improvement via telemedicine . although payer organizations have been reluctant to accept telemedicine due to uncertainties in its efficacy , an increasing body of literature demonstrates that telemedicine may be as effective ( and in some cases superior to ) the current standard of care in the treatment of chronic diseases such as diabetes , hypertension , and aids . obstructive sleep apnea ( osa ) is present in 420% of adults ( depending on the study and the operational definition ) and remains underdiagnosed [ 7 , 8 ] . osa is often comorbid with other chronic conditions such as heart disease and hypertension , and requires long - term management . home sleep testing devices are increasingly common , and even telemetric continuous positive airway pressure ( cpap ) titration has been shown to be effective . patients receiving in - person visits or video visits were equivalent in terms of satisfaction and treatment adherence . we undertook the current study to answer three important questions regarding patient perceptions of in - person versus telemedicine forms of patient care in an academic sleep disorders clinic population as follows : ( 1 ) what are the barriers to in - person clinic visits ? we administered a brief survey , consisting of 14 multiple choice questions and one open - ended free text question , to patients seen in the sleep disorders clinic at our institution between 20092011 ( see supplemtaery material at doi:10.5402/2012/135329 ) . the survey included questions pertaining to waiting times to be seen at primary care and specialist offices as well as communication practices , frequency , and preferences in regards to email , phone , video chat , and patient gateway ( see supplemtaery material ) . patient gateway is a local service offered by partners health care system ( massachusetts general hospital and brigham and women 's hospital ma , usa ) that allows patients to electronically communicate with their doctors through a secure platform . we included adult sleep disorder patients who had been seen by one of two board - certified sleep neurologists at massachusetts general hospital in boston , massachusetts for a variety of complaints ( the majority being for sleepiness , sleep apnea , and insomnia evaluations ) . this is an academic center serving a range of patient demographics ; most reside in massachusetts , with a minority traveling from neighboring states . we sent an email survey to patients if they had a viable email address on file ( n = 282 ) . an additional 156 patients , who did not provide an email address during routine clinical intake , were mailed a hardcopy of the questionnaire . these values do not include the 18 patients with invalid emails and the four patients for whom the hardcopy survey was returned due to invalid mailing address . the partners research committee determined that this study was exempt from institutional review board approval ; patients provided implied consent by their participation in the survey . study data were collected and managed using redcap ( research electronic data capture ) software developed at vanderbilt university , and hosted at massachusetts general hospital . redcap is a secure , web - based application designed to support data capture for research studies . redcap provides an intuitive interface for validated data entry ; audit trails for tracking data manipulation and export procedures ; automated export procedures for seamless data downloads to common statistical packages ; procedures for importing data from external sources . survey responses were analyzed with prism ( graphpad software , la jolla , ca , usa ) . the kruskall - wallis nonparametric test ( with dunn 's post hoc test ) was used for group comparisons . for some survey questions , we combined certain answers in order to preserve the ordinal nature of the list for statistical analysis . for example , the question regarding comfort with video telemedicine , we combined the not comfortable with not sure , which is a conservative change regarding estimation of opinion regarding video . to address the question of whether nonresponders differed in terms of age or sex , we selected a random sample of 50 nonresponders from the email cohort and the mail cohort . we found no significant difference in response rate according to sex for either mail or email surveys ( p > 0.05 , fisher 's exact test ) . for the email survey cohort , we found no difference in age between responders and the sample of nonresponders . however , for the mailed survey cohort , the respondents were older than nonresponders ( 62 versus 51 years of age ; p < 0.05 , anova with bonferroni correction ) . the response rate from those who were emailed was similar to those who received the survey by postal mail ( 30% versus 24% , p > 0.2 , fisher 's exact test ) , yielding an overall response rate of 28% ( table 1 ) . 83% of patients reported waiting under three months to be seen for their initial sleep consultation and for their follow - up visits ( figure 1(a ) ) . the waiting time for follow - up appointments with primary care physicians was similar , except that waiting times of less than 1 month were more common . satisfaction with sleep clinic waiting times was modest , with 41% of patients reporting they were either somewhat satisfied or not satisfied ( figure 1(b ) ) . patients indicated several important concerns regarding routine in - person clinic visits ( table 2 ) . the most common challenges for face - to - face appointments were cost of parking ( 44% ) , time away from work / school ( 34% ) , cost of gas ( 26% ) , and requiring family or other support to travel ( 19% ) . about 28% of patients reported they were sometimes or frequently late for in - person appointments ( data not shown ) . telephone contact was the most common form of telemedicine , with a large majority ( 89% ) of respondents employing this method at least 1 - 2 times per six months ( figure 2 ) . more than half of respondents reported contacting their doctors by email , with the most common frequency being 1 - 2 times per six months . about 30% of respondents reported using the encrypted electronic e - mail communication platform ( see methods ) . only 20% of those surveyed reported using a health diary as part of their care plan ( data not shown ) . despite the lack of experience using video for clinical purposes , the majority of patients ( 63% ) reported being very comfortable or willing to try this method of telemedicine ( figure 3(a ) ) . more than half ( 54% ) of respondents indicated they would be willing to pay a copay for a video appointment , and not surprisingly , they felt $ 10 or $ 25 was preferable to a $ 50 copay ( figure 3(b ) ) . of the 14% of respondents who were uncomfortable with video communication , the two most common reasons were that in - person visits feel more natural and that the doctor might need to perform an examination ( table 3 ) . only 13% of respondents felt video technology was too difficult or reported that they did not have a computer or internet connection . we tested whether respondent comfort with video telemedicine was related to age , sex , internet availability , willingness to pay a copay , or availability of an email address on file . uncertain and not comfortable responses ( see figure 3(a ) ) , a conservative assumption that allowed the responses to remain ordered for statistical testing ( nonparametric anova ) . there was no difference in comfort level with video telemedicine by age , sex , or by method of survey response ( mail versus email ) . of those who reported being uncomfortable or unsure about video telemedicine , 11% reported lacking a computer or internet access as a reason , and only 2% indicated it was too difficult . this subgroup of responders was also less willing to pay a copay ( p < 0.001 ) . this survey study indicates that a substantial portion of patients seen in an academic sleep disorders clinic are willing to consider video telemedicine as an option for their care . patients identified several practical barriers to in - person visits , including cost and inconvenience . their main concerns regarding video visits , including feeling less natural and the need for physical examination , are already inherent in telephone communication , which they reported commonly utilizing . these results are encouraging for the development of video appointments in sleep medicine and is consistent with previous evidence that patients find tele - consultation equivalent to in - person consultation [ 14 , 15 ] , as well as a forward - looking editorial suggesting the importance of telemedicine in the care of sleep disorders . indirect benefits include avoidance of barriers to in - person visits , such as the time and cost associated with travel or missed work . to decrease wait times by changing the medium of communication from in - person to virtual , either the visit duration would have to be shorter or the filling of cancellations would have to be more efficient . regarding the sense that video chat does not seem as natural as in - person visits , it is worth mentioning that phone and email , which are arguably even less natural - feeling , were commonly utilized by this cohort . it is possible that sentiments towards video communication will evolve to be even more accepted by patients as it becomes commonplace . regarding the concern that the doctor might need to perform some physical examination , in sleep medicine it may be that certain stable patients could be adequately assessed without performing a physical exam that requires the physician to be present in the same room . in addition , certain elements of the examination such as weight or blood pressure could be performed at home or at a local health clinic . additionally , many patients using video visits reported the absence of a physical exam to be acceptable and gave the experience high satisfaction scores . although we did not assess this aspect directly , one respondent did express privacy concerns in the free text field . the majority of patients were willing to pay either $ 10 or $ 25 per video appointment . with the increasing evidence of the treatment efficacy and patient support of telemedicine in a variety of medical settings , sleep medicine may experience similar benefits in terms of their chronic management . the extent to which third party payers might reimburse for virtual visits in sleep medicine remains uncertain but there are emerging mechanisms for supporting these types of visits financially if they can be shown to lower costs . physician acceptance of telemedicine incorporates personal preference , prior experience , reimbursement potential , and demonstration of improved or equivalent satisfaction and outcomes compared to in - person visits . telephone contact with patients between clinic visits is employed across many specialties , especially for low - level decision making . the majority of physicians do not charge for telephone services , yet most are in agreement that compensation for their time is appropriate , whether in person or electronically . concerns about telephone - based care from the physician standpoint include limited access to the patient 's health record , legal concerns about advice delivered in this setting , and challenges of documentation . in specialties with particularly nuanced and complex examinations , such as neurology , telemedicine for diagnostic purposes neurologists were also reported to universally feel that they were able to appropriately communicate management advice to their patients by video . in sleep medicine , the extent to which physical examination influences chronic decision making in common disorders such as insomnia and sleep apnea remains untested . in regards to the rising costs of health care , telemedicine may be a promising solution , as many studies have shown scheduling video chat appointments results in cost - savings for patients and hospitals . in one economic review of video telemedicine , 22 of 36 studies reported this method to be more cost effective than standard office appointments . video appointments could address a variety of the burdens associated with in - person visits listed by patients in this survey . for example , the time , cost , and inconvenience associated with travel could be ameliorated by a video visit option ( table 2 ) , as has been reported for example in pediatrics and oncology . in addition , the decreased utilization of scarce outpatient practice space due to offloading to telemedicine - based practice could free up space for patients requiring in - person consultation . telemedicine visits might also be useful for deciding which chronic patients require further in person care . sleep medicine may be an ideal specialty in which video visits could accommodate routine chronic follow - up appointments . patient - specific data , such as diaries for the insomnia patient and cpap machine downloads for the osa patient , could be reviewed at such visits , as could routine challenges with medications or equipment . seeing the patient in their home environment may assist the clinician in more accurately diagnosing reasons for sleep dysfunction including ill - fitting equipment , certain aspects of poor sleep hygiene , or an overly illuminated bedroom . in a recent editorial , telemedicine was proposed as an important method to approach the ongoing challenges of cost - effective and outcomes - oriented chronic care of patients with sleep disorders . one study recently reported the successful use of video telemedicine in osa patients , with similar treatment adherence and satisfaction levels compared to those with in - person visits . this opportunity may extend to management of other chronic diseases in which evaluations and decision making may be considered using only remotely obtainable information . the main limitations of this study include the 28% response rate and the single - center population . nevertheless , our findings highlight several important points and lay the groundwork for future development of sleep telemedicine . patients experience multiple barriers to in - person visits that could be circumvented with virtual visits , and the majority of respondents were willing to pay a copay for a video visit despite none of the respondents having any personal experience with video telemedicine . chromosome 22q13.3 deletion syndrome is a well - recognized cause of global developmental delay ( gdd ) [ 1 , 2 ] while duplication of the same chromosome is a rare occurrence [ 35 ] . the presence of both abnormalities in the same family has never been reported , to our knowledge . we report the presence of 22q13.3 deletion and 22q13.3 duplication in two siblings , with the mother carrying an inversion of chromosome 22 ( 46 , xx , inv ( 22 ) ( p13q13.32 ) ) a 6 year - old male was noted in infancy to have mild global developmental delay without dysmorphic features . his 4 year - old brother was noted in early infancy to have severe global developmental delay and dysmorphic features related to 22q13.3 deletion to the terminus . the mother 's inversion may rearrange to form 22q duplication or deletion when passed on to children . the chance of a child born with a chromosome imbalance is as high as 50% . he had calcaneo - valgus deformity of the right foot which improved with manipulation with each diaper change . at 9-month - old , his mother noticed that he would not engage with her and would stare into space . he was referred to a neurodevelopmental pediatrician at age 18 months because all developmental milestones were delayed . his height and weight were at 50th percentile , and fronto - occipital circumference ( foc ) was at 98th percentile ( large head ) . neuropsychological evaluation revealed significant difficulty in social skills and adaptive functioning , consistent with a diagnosis of pervasive developmental disorder nos . it further revealed that receptive language was better than expressive language skills . a metabolic disorder screen ( plasma for amino acids , urine for organic acids , smith - lemli opitz , thyroid ) and fragile - x screening were normal . chromosomal analysis revealed 46 , xy , rec ( 22 ) dup ( 22q ) inv ( 22 ) ( p13 ; q13.32 ) mat ( figures 1 and 2 ) . jc is the younger sibling who was born by normal vaginal delivery , after a term gestation , when his mother was 38 yrs old . his birth weight was 9 lb . 3 oz . he had transient tachypnea of the newborn which responded to oxygen administration . jc was referred to a neurodevelopmental pediatrician at the age of 13 mos . because of delayed language and motor development . height and weight were in the 90th percentile , and foc was between 70 and 90th percentile . he had downslanting palpebral fissures , fleshy hands , dysplastic toe nails , and clinodactyly . he has generalized coarse facial features , broad nasal bridge , relatively flat midface , and prominent chin and ears . he has heat intolerance ( because of decreased perspiration ) , a high pain threshold , and a wobbly gait ( because of hypotonia ) . behavior characteristics included mouthing and chewing of nonfood items and autistic - like behavior and affect , typical of the 22q13.3 deletion syndrome . newborn genetic screening ( amino academia , congenital adrenal hyperplasia , congenital hypothyroidism , smith - lemli opitz , galactosemia , hemoglobinopathy ) was normal . chromosomal analysis revealed 46 , xy , rec ( 22 ) dup ( 22p ) inv ( 22 ) ( p13q13.32 ) mat ( figures 1 and 2 ) . medication management for his disruptive behaviors was strongly recommended , but the family refused this option because of the potential sideeffects . the mother ( hc ) has an inversion in one of her number 22 chromosomes : 46 , xx , inv(22 ) ( p13 ; q13.32)indicating that one of the mother 's number 22 chromosomes contains a large piece that is inverted or flipped backwards ( figure 3 ) . her mother 's first paternal cousin has an intellectual disability but went on to have 3 children , and one of them is intellectually disabled . the father ( tc ) has entirely normal chromosomes . 22q13 would not be considered a classic hot spot for genomic rearrangements , which are generally regions flanked by highly homologous segmental duplications which predispose to nonhomologous allelic recombination during meiosis in one of the parental lines , yielding identical and recurrent deletions or the reciprocal duplication . although such individuals are completely healthy , a certain proportion of their gametes will contain a rearranged form of the inverted 22 chromosome . a parent with an inversion or a balanced translocation could transmit the unbalanced form of the translocation to the offspring , or deletions and duplications may occur through recombination between the normal allele and the inverted allele . a child with this unbalanced rearrangement would be expected to have developmental delay and possible birth defects . this rearrangement can be in the form of deletion or duplication of the chromosomal segment in question . in the remaining off - springs , where the rearrangement remains balanced alternately , the mother may pass on her normal number 22 chromosome , and the child will be normal . cc , the older sibling ( 46 , xy , rec ( 22 ) dup ( 22q ) inv ( 22 ) ( p13 ; q13.32 ) mat , ) has no dysmorphic features , yet has significant developmental delay . the 22 chromosome duplications are very rare , and the ones cited in the literature have breakpoints on 22q12 rather than 22q13 . jc , with monosomy of 22q13.3 , has findings typical of 22q13.3 deletion syndrome patients including global developmental delay , generalized hypotonia , absent or severely delayed speech , and normal or accelerated growth . the true prevalence of 22q13.3 deletion may be underestimated because routine karyotyping may only detect a fraction of the cases . when this condition is suspected based on clinical features or there is a history of mild gdd in an older sibling , early genetic testing should be instituted employing new and more assertive methods like genomic microarray and fluorescent in situ hybridization ( fish ) . it is worth noting that our case ( jc ) acquired the deletion from his mother . in addition , jc also received music therapy , picture exchange communication , and sign language instruction . it is believed that these interventions contributed to improving behavioral issues ( autistic features ) in jc and global developmental delay in both . to facilitate communication and decrease frustration within the family , all family members should be encouraged to learn sign language [ phelan , personal communication ] . as is true for most chromosomal abnormalities , and as was done with the presented cases , individuals with 22q13.3 deletion or duplication should also undergo base - line investigations like renal ultrasound ( for renal cysts and other structural problems ) , echocardiogram ( for septal defects ) , eeg ( for seizure activity ) , audiology tests ( to r / o hearing deficits ) , and brain imaging studies ( arachnoid cysts , etc . ) . as these individuals are expected to have a reasonably normal life expectancy [ phelan , personal communication ] , they should receive the recommended health maintenance preventive services ( pap smears , colon cancer screening , prostate exam , cholesterol check , etc . ) . some screening tests like colon cancer screening may need to be expedited , as there may be an increased predisposition towards colon cancer . amniocentesis for prenatal testing in pregnancies at increased risk for chromosomal abnormalities may not be diagnostic , as in jc 's case [ dr . grati , european genetics conf . 2008 ] , and fish , chorionic villus sampling , or micro - array testing should be considered , as they are more specific . behavioral interventions and medication management such as atypical neuroleptics are often recommended to control behavior problems .

telemedicine is an increasingly recognized option for cost - effective management of chronic conditions . we surveyed sleep clinic patients about their experiences and preferences regarding different forms of telemedicine . adult sleep clinic patients seen between 2009 and 2011 received a brief survey either by postal mail ( n = 156 ) or , for those with an available email address , electronically ( n = 282 ) . the overall response rate was 28.1% ( n = 123 responses ) , with email response rates being higher than postal mail responses . the most commonly reported barriers to in - person physician visits were parking cost ( 44% ) , time away from work / school ( 34% ) , and cost of gas ( 26% ) . whereas 89% of respondents indicated using telephone and 55% of respondents indicated using email to communicate with providers , none reported experience with video telemedicine . despite this lack of experience , over 60% reported feeling comfortable or willing to try it . of those who were uncomfortable about video telemedicine , the two main reasons were that in - person visits feel more natural ( 48% ) and that the doctor might need to perform an examination ( 24% ) . more than half of respondents reported willingness to pay a copay for a video visit . video telemedicine represents a feasible option for chronic sleep disorders management . chromosome 22q13.3 deletion syndrome is a well - recognized cause of global developmental delay , while duplication of the same chromosome is a rare occurrence . the presence of both abnormalities in the same family has never been reported , to our knowledge . we report a rare occurrence of 22q13.3 duplication and 22q13.3 deletion in siblings , as a consequence of a mother 's inversion on her 22nd chromosome ( p13;q13.32 ) . a 6 year old male was noted in infancy to have mild global developmental delay without dysmorphic features . his genetic testing revealed he had 22q13.3 duplication to the terminus . his 4 year old brother was noted in early infancy to have severe global developmental delay and dysmorphic features related to 22q13.3 deletion to the terminus . their mother had a long inversion on her 22nd chromosome . genetic tests for their father and eldest brother were unremarkable . the mother 's inversion may rearrange to form 22q duplication or deletion when passed on to children . the chance of a child born with a chromosome imbalance is as high as 50% .

in this study , we retrospectively selected 75 cases of formalin - fixed , paraffin - embedded hair - bearing adult skin samples between 2002 and 2009 at asan medical center . samples were taken from the margins of routine surgical procedures such as excisional biopsy or excision for benign / malignant skin lesions that were diagnosed as following : various inflammatory lesions with parakeratosis ( n=16 ) , keratinocytic tumors ( n=11 ) , benign tumors with apocrine and eccrine differentiation ( n=14 ) , malignant tumors with follicular differentiation ( n=1 ) , benign tumors with follicular differentiation ( n=17 ) , and tumors with sebaceous differentiation ( n=12 ) . the rest were retrieved from mature cystic teratoma ( n=2 ) and normal scalp tissues biopsied for therapeutic reasons ( n=2 ) . to analyze cd99 expression patterns in fetal skin , gestational ages of the collected cases were 16 , 17 , 19 ( 2 cases ) , 20 , 25 , 26 , and 28 weeks . all tissues were immediately fixed in 10% buffered formalin , and then processed in paraffin wax using standard procedures . sections were serially cut into 4-m sections and stained with hematoxylin and eosin for examination . the study protocol was approved by the institutional review board ( project number 2009 - 440 ) of asan medical center . when multiple blocks were available in a single case , one block containing representative tissue was selected . ihc staining for cd99 was performed using a benchmark xt autoimmunostianer ( ventana medical systems , tucson , az , usa ) according to the manufacturer s instructions and using the reagents supplied with the kit . in brief , 4-m sections were mounted on silanized charged slides , dried for 10 minutes at room temperature and then further dried for 20 minutes at 65c . after deparaffinization , heat - induced epitope retrieval using standard cell conditioning solution 1 was performed for 24 minutes . subsequently , primary anti - cd99 ( 1:100 , clone dn16 , dinona , seoul , korea ) was applied by an automated immunostaining system with the ultraview dab detection kit ( ventana medical systems ) . analysis of whole tissue section slides stained for cd99 by ihc was performed under a light microscope as previously described [ 14 - 16 ] . the results of cd99 immunostaining were classified into the following groups : consistently complete and strong membrane staining was assigned a value + + , weak to moderate membrane staining was assigned a value of + , variable membrane staining was assigned a value of + / , and no staining was assigned a value of . occasional cytoplasmic staining was minimal and weak . for immunofluorescence ( if ) staining , 4-m sections were incubated with mouse anti - cd99 ( 1:200 , clone dn16 , dinona ) and rabbit anti - cd34 ( 1:10 , # ab64480 , rabbit polyclonal , abcam , cambridge , uk ) for 60 minutes at room temperature . sections were then incubated with tetramethylrhodamine - conjugated host anti - mouse igg antibody and fluorescein isothiocyanate - conjugated host anti - rabbit igg antibody for 60 minutes at room temperature . sections incubated with the appropriate isotype control primary antibodies and fluorescently labeled secondary antibodies were utilized as necessary . results were analyzed using confocal microscopy ( leica tcs_np / sp , leica microsystems , mannheim , germany ) . in this study , we retrospectively selected 75 cases of formalin - fixed , paraffin - embedded hair - bearing adult skin samples between 2002 and 2009 at asan medical center . samples were taken from the margins of routine surgical procedures such as excisional biopsy or excision for benign / malignant skin lesions that were diagnosed as following : various inflammatory lesions with parakeratosis ( n=16 ) , keratinocytic tumors ( n=11 ) , benign tumors with apocrine and eccrine differentiation ( n=14 ) , malignant tumors with follicular differentiation ( n=1 ) , benign tumors with follicular differentiation ( n=17 ) , and tumors with sebaceous differentiation ( n=12 ) . the rest were retrieved from mature cystic teratoma ( n=2 ) and normal scalp tissues biopsied for therapeutic reasons ( n=2 ) . to analyze cd99 expression patterns in fetal skin , gestational ages of the collected cases were 16 , 17 , 19 ( 2 cases ) , 20 , 25 , 26 , and 28 weeks . all tissues were immediately fixed in 10% buffered formalin , and then processed in paraffin wax using standard procedures . sections were serially cut into 4-m sections and stained with hematoxylin and eosin for examination . the study protocol was approved by the institutional review board ( project number 2009 - 440 ) of asan medical center . all hematoxylin and eosin stained slides were reviewed to ensure quality . when multiple blocks were available in a single case , one block containing representative tissue was selected . ihc staining for cd99 was performed using a benchmark xt autoimmunostianer ( ventana medical systems , tucson , az , usa ) according to the manufacturer s instructions and using the reagents supplied with the kit . in brief , 4-m sections were mounted on silanized charged slides , dried for 10 minutes at room temperature and then further dried for 20 minutes at 65c . after deparaffinization , heat - induced epitope retrieval using standard cell conditioning solution 1 was performed for 24 minutes . subsequently , primary anti - cd99 ( 1:100 , clone dn16 , dinona , seoul , korea ) was applied by an automated immunostaining system with the ultraview dab detection kit ( ventana medical systems ) . analysis of whole tissue section slides stained for cd99 by ihc was performed under a light microscope as previously described [ 14 - 16 ] . the results of cd99 immunostaining were classified into the following groups : consistently complete and strong membrane staining was assigned a value + + , weak to moderate membrane staining was assigned a value of + , variable membrane staining was assigned a value of + / , and no staining was assigned a value of . occasional cytoplasmic staining was minimal and weak . for immunofluorescence ( if ) staining , 4-m sections were incubated with mouse anti - cd99 ( 1:200 , clone dn16 , dinona ) and rabbit anti - cd34 ( 1:10 , # ab64480 , rabbit polyclonal , abcam , cambridge , uk ) for 60 minutes at room temperature . sections were then incubated with tetramethylrhodamine - conjugated host anti - mouse igg antibody and fluorescein isothiocyanate - conjugated host anti - rabbit igg antibody for 60 minutes at room temperature . sections incubated with the appropriate isotype control primary antibodies and fluorescently labeled secondary antibodies were utilized as necessary . results were analyzed using confocal microscopy ( leica tcs_np / sp , leica microsystems , mannheim , germany ) . to examine the expression pattern of cd99 in epidermal keratinocytes , immunostaining for cd99 was performed on nonpathologic adult skin samples ( fig . 1a ) . in the epidermis , cell membranes in the basal cell layer were strongly stained ( fig . 1b ) , whereas the keratinocytes in the prickle cell layer just above the basal cell layer showed markedly reduced staining and cells in the granular and hornified layers did not express cd99 . we also identified intense cd99 expression in langerhans cells in the epidermal layer ( fig . infundibula of hair follicles possessed a cd99 expression pattern similar to that of the epidermis . 1c ) at the bulges , and basal cells of the follicular ors were also strongly stained ( fig . other subpopulations of hair follicles showed varying immunoreactivity . when observing cd99 staining in the central shaft of hair follicles , the cuticle was negative for cd99 , the lower irs was weakly positive , and the ors was strongly positive ( fig . to flat cell - lined apocrine glands were negative for cd99 , but relatively tall cell - lined apocrine glands were positive for cd99 . the apical luminal surfaces of both flat and tall cell - lined glands were cd99-negative ( fig . secretory coils of eccrine sweat glands are composed of outer clear cells and inner cuboidal cells ; the outer clear cells were immunonegative and scattered inner cuboidal cells were immunopositive for cd99 ( fig . the excretory duct is composed of two - layered cuboidal cells , and both layers were immunopositive for cd99 ( fig . the apical luminal surfaces of inner eccrine cells and inner excretory ducts also lacked cd99 expression ( fig . 2b , c ) . in the sebaceous glands , basal cells surrounding the sebaceous alveolus were cd99 immunopositive , but mature sebocytes containing central lipid droplets were immunonegative for cd99 ( fig . cells that were positive for cd99 immunostaining in normal adult epidermis and skin appendages are summarized in table 1 . eight developing fetal human skin samples were obtained from aborted fetuses of 1628 gestational weeks . in early stages , fetal skin is called periderm and is composed of 23 epidermal cell layers . at 16 weeks of gestation , the periderm , including the epidermal basal layer and hair follicle germ , was negative for cd99 , whereas mesenchymal cells around the hair germ were strongly positive for cd99 ( fig . , the fetal epidermis and skin appendages start to resemble normal adult skin ; basal cell layer , intermediate cell layer , hair follicles , sweat glands , and sebaceous glands appear at this stage . cd99 was expressed in the epidermal basal cell layer , hair bulge , ors , and dermal papilla ( fig . fetal skin is histologically similar to adult skin , showing cd99 immunoreactivity patterns identical to those of adult skins ( fig . 3c , d ) . in our study , cd99 staining was particularly strongly positive in immature epidermal basal cells and cells at the bulge . we therefore compared cd99-positive subpopulations of normal epidermis and skin appendages to cd34-positive subpopulations that have been reported as cutaneous precursor cells using double - if . we focused on expression patterns of these markers in the basal cells of epidermis and hair follicles . in the adult epidermis and skin appendages , cd99 and cd34 expression partially overlapped at the lower ors ( fig . c ) . in the 20-week - old fetal skin , cd99 and cd34 were not co - expressed in the basal cells of epidermis ( fig . 5c ) , or ors ; however , mesencymal cells of dermal papilla showed co - expression ( fig . to examine the expression pattern of cd99 in epidermal keratinocytes , immunostaining for cd99 was performed on nonpathologic adult skin samples ( fig . 1a ) . in the epidermis , cell membranes in the basal cell layer were strongly stained ( fig . 1b ) , whereas the keratinocytes in the prickle cell layer just above the basal cell layer showed markedly reduced staining and cells in the granular and hornified layers did not express cd99 . we also identified intense cd99 expression in langerhans cells in the epidermal layer ( fig . infundibula of hair follicles possessed a cd99 expression pattern similar to that of the epidermis . 1c ) at the bulges , and basal cells of the follicular ors were also strongly stained ( fig . other subpopulations of hair follicles showed varying immunoreactivity . when observing cd99 staining in the central shaft of hair follicles , the cuticle was negative for cd99 , the lower irs was weakly positive , and the ors was strongly positive ( fig . secretory apocrine glands showed variable cd99 expression . relatively thin to flat cell - lined apocrine glands were negative for cd99 , but relatively tall cell - lined apocrine glands were positive for cd99 . the apical luminal surfaces of both flat and tall cell - lined glands were cd99-negative ( fig . secretory coils of eccrine sweat glands are composed of outer clear cells and inner cuboidal cells ; the outer clear cells were immunonegative and scattered inner cuboidal cells were immunopositive for cd99 ( fig . the excretory duct is composed of two - layered cuboidal cells , and both layers were immunopositive for cd99 ( fig . the apical luminal surfaces of inner eccrine cells and inner excretory ducts also lacked cd99 expression ( fig . basal cells surrounding the sebaceous alveolus were cd99 immunopositive , but mature sebocytes containing central lipid droplets were immunonegative for cd99 ( fig . cells that were positive for cd99 immunostaining in normal adult epidermis and skin appendages are summarized in table 1 . eight developing fetal human skin samples were obtained from aborted fetuses of 1628 gestational weeks . in early stages , fetal skin is called periderm and is composed of 23 epidermal cell layers . at 16 weeks of gestation , the periderm , including the epidermal basal layer and hair follicle germ , was negative for cd99 , whereas mesenchymal cells around the hair germ were strongly positive for cd99 ( fig . , the fetal epidermis and skin appendages start to resemble normal adult skin ; basal cell layer , intermediate cell layer , hair follicles , sweat glands , and sebaceous glands appear at this stage . cd99 was expressed in the epidermal basal cell layer , hair bulge , ors , and dermal papilla ( fig . fetal skin is histologically similar to adult skin , showing cd99 immunoreactivity patterns identical to those of adult skins ( fig . in our study , cd99 staining was particularly strongly positive in immature epidermal basal cells and cells at the bulge . we therefore compared cd99-positive subpopulations of normal epidermis and skin appendages to cd34-positive subpopulations that have been reported as cutaneous precursor cells using double - if . we focused on expression patterns of these markers in the basal cells of epidermis and hair follicles . in the adult epidermis and skin appendages , cd99 and cd34 expression partially overlapped at the lower ors ( fig . c ) . in the 20-week - old fetal skin , cd99 and cd34 were not co - expressed in the basal cells of epidermis ( fig . 5c ) , or ors ; however , mesencymal cells of dermal papilla showed co - expression ( fig . the aim of the current study was to examine cd99 expression in normal human skin and skin appendages ; no such study has been performed to date . we demonstrated that cd99 was strongly expressed in the immature epidermal basal cells , but the fully differentiated granular layer did not show cd99 positivity in normal adult skin . this result is consistent with the findings of a previous study , in which cd99 expression was evaluated in a large series of cutaneous melanoma samples and was described briefly as a crisp membranous staining on normal epidermal basal cells . our study showed that in skin appendages , cd99 showed differential immunoreactivity in each subpopulation . bulge cells , basal cells of the ors , and eccrine inner cuboidal cells were consistently and strongly positive . however , cd99 expression was variably positive in apocrine secretory glands . relatively tall cells with luminal decapitation tended to be cd99-positive , but thin to flat cells without decapitation tended to be negative . analysis of cd99 expression was also performed during different developmental stages of fetal skin . in fetal skin , peridermal cells persist until about week 21 and gradually disappear as stratum corneum . in the early stages of epidermal development , mitotic activity occurs in all the epidermal layers ; however , when differentiation begins , mitotic activity becomes restricted to the cells of the basal layer . angiotensin converting enzyme ( ace ) , keratin 19 , 1-integrin , and p63 were used as stem cell markers in examining the various developmental stages of fetal skin by double if . the expression of ace , 1-integrin , keratin 19 , and p63 was seen in all epidermal layers of the developing fetal skin at 1120 weeks of gestation . from 21 weeks of gestation onward , their expression was mainly confined to the basal layer of epidermal cells . in our study , cd99 was not seen in the periderm at 16 weeks of gestation but was consistently expressed at 19 weeks of gestation . based on this result , we suggest that cd99 expression precedes the expression of ace , 1-integrin , keratin 19 , and p63 in the fetal basal layer . based on the particularly strong cd99 expression in immature thymic t - lineage cells , tonsillar lymphoid progenitor cells , and subventricular zone of fetal brain ( c .- s . park , unpublished data ) , we attempted to correlate cd99 expression with immature subpopulations of epidermis and appendages . there is evidence for populations of stem cells located at the basal layer of the epidermis and the hair follicle bulge . cd99 is also strongly expressed in the bulge . according to previous reports that analyzed expression patterns of various precursor markers , cd34 is more specific for the peripheral layer of the ors , cytokeratin ( ck ) 15 for bulge and epidermal basal cells , nestin for inner portions of the ors , ck19 for basal keratinocytes in the upper and lower third of the ors , p63 for the basal / suprabasal layer , hair matrix , and ors , and cd200 for the bulge [ 20 - 24 ] . we showed that cd34 , which was thought to have similar expressions patterns to cd99 , shows different expression by double if . in the present study , cd34 and cd99 were only co - expressed in the lower ors . therefore , we assumed that precursor cell subpopulations in the skin are more varied than previously thought . in conclusion , this study examined cd99 expression in normal adult and fetal skin . cd99 is strongly expressed in the immature epidermal basal cells in the bulge and basal cells of the hair follicle ors . these findings suggest that cd99 may carry out a specific function in the structures where it is expressed . therefore , we present that cd99 is a unique and previously unreported marker of epidermis and its appendages . future studies should focus on functions of cd99 in the epidermis and its appendages and investigate its feasibility as a novel target for the treatment of dermatologic lesions . glaucoma remains the third or fourth most common cause of blindness in different regions around the globe . since this is a treatable disease , early diagnosis and adequate follow - up are gaining importance . also , direct and indirect economic burdens tend to increase for governments with patients extended lifetime expectancy and complexity of disease stage . so far standard automated perimetry ( sap ) is still considered the gold standard method for function analysis even though defects are only detectable after substantial cell loss . since its introduction , optical coherence tomography ( oct ) has turned into a fundamental tool in the evaluation of a variety of different retinal diseases , in particular primary open angle glaucoma ( poag ) . thinning of the peripapillary retinal nerve fiber layer ( rnfl ) and full macular thickness ( mt ) have been largely used in poag evaluation [ 3 , 4 ] . in addition , recent improvements in oct technology ( i.e. , spectral oct and software analysis ) not only increased image resolution but also allowed customized analysis of the individual retinal layers . glaucoma damage is primarily related to the ganglion cells . thus oct retinal layer segmentation allows us to directly analyze the ganglion cell layer separately and thereby look directly at the site of damage . indeed , ganglion cell - inner plexiform layer ( gcipl ) segmentation can identify changes and correctly diagnose glaucoma with a similar sensitivity as the rnfl or optic nerve head ( onh ) parameters [ 68 ] . thus segmentation may allow a more sensitive structure - function correlation in different stages of disease . although the increased number of oct manufacturing companies may contribute positively to price competition as well as hardware and software improvements , it also leads to variability in measurements and analysis methodology . in this study , we determined if the brand of the spectral - domain oct used and their respective segmentation programs influence structure - function analysis differently . to the best of our knowledge , this is the first study to compare two different oct systems and their respective macula layer segmentation software and associate them with sap in structure - function analysis . the study protocol was approved by the ethics committee of the university of basel , and informed consent was obtained from all participants before the examination . all procedures followed the tenets of the declaration of helsinki . the inclusion criteria included a visual acuity of 0.8 or better , and a refractive error between 6 diopters of hyperopia or myopia . all glaucoma patients had a cup - to - disc ratio of at least 0.5 and a localized thinning of the neuroretinal rim on oct ( cirrus ) corresponding to the fundus examination . the oct thinning should have at least one red sector or two yellow sectors on the thickness map ( less than 1% and 5% of the normal population , resp . preperimetric glaucoma was defined by the presence of optic nerve abnormalities consistent with glaucoma and a normal visual field as tested with sap . other glaucoma patients had to present a reproducible glaucomatous visual field defect on at least three examinations with a mean defect ( md ) higher than 2.0 db and/or a squared root of loss variance ( slv ) over 2.5 db . individuals with previous ocular surgery , systemic diseases , or regular use of medications that could influence the eye ( e.g. , antidepressant , chloroquine ) were excluded from the study . the right eye was included in the study , if it did not fulfill any exclusion criteria . when not possible , visual field examination was performed at a maximum interval of 7 months from oct examination . technical details from the two different commercially available oct instruments used are displayed in table 1 . the pupil of the study eye was dilated with a solution of tropicamide 0.5% and phenylephrine 1% ( spital - pharmazie usb , switzerland ) before examination . oct images were obtained in cirrus using the fast macular cube protocol 512 218 ( 128 horizontal scan lines each composed of 512 a - scans , cirrus sd - oct , carl zeiss , usa ) , and the fast volume scan in spectralis hra + oct ( 25 section scans and 26 art frames , heidelberg engineering , inc . , heidelberg , germany ) . both instruments have a scan area of 6 6 mm and macular retinal thickness is calculated in microns in an area correspondent to the early treatment diabetic retinopathy study ( etdrs ) grid . the mt values used in this study corresponded to the 1 and 3 mm circles of the etdrs grid . gcipl thickness is calculated by cirrus software in the area of an elliptical annulus with a 2.0 mm vertical and 2.4 mm horizontal radius , excluding a central elliptical area ( 0.5 mm vertical and 0.6 mm horizontal radius ) that corresponded to the foveola . according to studies of human retina , the highest density of ganglion cells occurs in this area . as spectralis software ( version 6.0.3 ) uses the etdrs grid also for gcipl thickness calculation , values in the 3 mm circle were averaged , excluding the 1 mm circle , and compared to cirrus ( figure 1 , top ) . cirrus software excluded the macular rnfl layer from the gcipl analysis while spectralis software calculated each retina layer separately ( figure 1 , bottom ) . therefore , in spectralis , only the layers of interest in this study ( ganglion cell and inner plexiform layers ) were added in a separate microsoft excel spreadsheet . the exclusion of rnfl in cirrus was based on the histologic observation that the macular gcipl layer presents less variation than the rnfl among normal individuals . differences between the octs are that while both allow for manual corrections of the macular thickness boundaries , only spectralis allows for manual correction of possible errors in gcipl segmentation . cirrus , but not spectralis , separately analyses the minimum value of gcipl thickness ( mgcipl ) measured within the areas analyzed . thus parameters included in this analysis were averaged mt and the gcipl from both octs , and in addition , their average after manual correction ( cmt and cgcipl ) in spectralis and the mgcipl value given in cirrus . all images included in this study had signal strength over 7 for cirrus and a quality score over 25 for spectralis ( limits considered as good / acceptable image quality for analysis , according to each instrument 's manual ) . standard automated perimetry was performed using an octopus perimeter ( octopus 101 , g2 program , haag - streit ag , switzerland ) . total field md ( mean defect ) values in db were included in the analysis . all sap exams used in this study were inside reliability parameters ( fixation loss < 33% , false - positive and false - negative rates < 25% ) . differentiation between glaucoma and controls within each measurement was assessed with a t - test and p values posteriorly adjusted with fdr ( false discovery rate ) . the predictive diagnostic performance for each parameter , that is , the ability to differentiate glaucoma from control , was assessed calculating the receiver operating characteristics ( roc ) curves and the respective area under the roc curve ( auc ) . a perfect predictive performance is represented by an auc of 1.0 which means that this parameter can differentiate glaucoma from control with 100% sensitivity and specificity , while an auc value of 0.5 means a prediction mostly influenced by chance . aucs from different octs were compared with the delong test . for prediction of structure - function relationship all calculations were adjusted to age . as oct values are linear values but sap are reported in db , a logarithmic value , oct values were transformed to logarithmic scale for better comparisons with sap . results were expressed as the regressive slope coefficients ( on log - scale ) with corresponding standard errors and p values . statistical analyses were performed using spss ( ibm spss statistics , version 22 ) , and the statistical package r ( version 3.0.2 ) . in this study , the mean age was 59.5 years ( sd 13.9 ) for the glaucoma group and 49.2 years ( sd 7.0 ) for the controls ( p = 0.013 ) . median md was 2.2 ( range : 0.417.0 ) db in poag ( including ppg ) and 0.2 ( range : 3.82.0 ) db in controls ( p = 0.024 ) . table 3 shows averages from mt and gcipl in both octs in the poag and control groups . spectralis and cirrus showed a significant difference between patients and controls in both mt ( p = 0.018 , p = 0.028 , resp . ) and gcipl ( p < 0.001 , both ) . manual correction of the software segmentation parameters in spectralis produced a significant difference between measurements in gcipl ( p < 0.05 ) but not in mt ( p = 0.715 ) . a total of 2 controls ( 12.5% ) and 7 patients ( 41.1% ) needed posterior manual correction of retina thickness segmentation . bland - altman analysis showed disagreement between octs in mt and gcipl values ( figure 2(b ) ) . on average , measurements with spectralis were thicker than with cirrus . for mt the difference was 21.64 m ( sd 4.5 ) before and 21.65 m ( sd 4.5 ) after manual correction ( p < 0.001 ) . for average gcipl thickness the difference was 9.8 m ( sd 5.4 ) before and 10.0 m ( sd 5.3 ) after manual correction ( p < 0.001 ) . with higher values measurements obtained with spectralis tended to differ more from those measured with cirrus . this difference increased when we compared spectralis averages before ( 14.1 m , sd 5.9 ) and after correction ( 14.4 m , sd 5.8 ) with mgcipl ( p < 0.001 ) . there was no significant difference between the age - adjusted aucs from mt in cirrus ( 0.798 ) and spectralis , before ( 0.801 ) and after ( 0.805 ) manual correction . this was also observed between octs for gcipl measurements : 0.879 in cirrus and 0.886 before and 0.886 after correction in spectralis . minimum gcipl value in cirrus had an auc of 0.930 ( table 3 ; figure 3 ) . mt and gcipl had a negative significant association with md ( p < 0.001 ) , in both poag ( p < 0.001 ) and controls ( p < 0.001 ) for cirrus and spectralis ( table 4 , figure 4 ) . the aim of this study was to compare thickness measurements between two commercially available octs using their respective segmentation programs and assess if the brand of spectral - domain oct used might influence structure function analysis differently in glaucoma . using two different spectral - domain octs , cirrus and spectralis , we observed that there is a significant difference in full macula and gcipl thickness measurements between machines . therefore measurements are not interchangeable . nevertheless , when assessing structure function relationship individually , all measurements from both machines demonstrated a statistically significant relationship with function measured by standard automated perimetry . further , age - adjusted aucs demonstrated that all measurements had a similar predictive performance and could correctly differentiate patients from controls . a literature review in pubmed using specific terms ( optical coherence tomography , glaucoma , ganglion cell , macula , thickness , and segmentation software ) did not reveal any other study which we could directly compare to this one . while we compared the entire area within the central 3 mm diameters , most studies either refer to the central 1 mm area [ 1116 ] or compare the areas within the etdrs sectors calculated by the respective octs ( i.e. , the superior , inferior , temporal , and nasal sectors ) [ 11 , 16 ] . here this is in accordance with a study from mylonas et al . , where spectralis macula thickness also showed the highest values , in the central retinal thickness ( crt , 1 mm diameter ) and individual sectors of the 3 mm area , in comparison to other octs ( including cirrus ) . though the study was conducted in neovascular age - related macular degeneration patients ( 28 individuals ) , its control group ( 10 individuals ) showed the same pattern . other studies found the same difference between cirrus and spectralis in crt [ 12 , 14 , 15 ] . the gcipl average in early glaucoma patients from these studies ( 69.7 m ) is comparable to our study average ( 68.5 m ) . a comparison of layer segmentation reproducibility was conducted at the iowa university using cirrus and their own segmentation software . here , the overall average from cirrus gcipl was reported as 70.0 m ( sd 11.4 ) in glaucoma , which also did not differ much from the patient group average in our study ( cirrus ) . recently , martinez - de - la - casa et al . demonstrated that , using spectralis layer segmentation software , macular rnfl thickness was the only parameter to differentiate healthy subjects from glaucoma suspects . we have not found a study comparing spectralis and cirrus segmentation software , most probably because spectralis software was made commercially available only recently . the clear difference in macula thickness between octs could be explained analyzing the specific retina boundaries established by each manufacturer . while the inner boundary is always the vitreoretinal interface , the outer retinal boundary varies between manufacturers . for cirrus the outer boundary corresponds to the level of the interdigitations of the external layers of the photoreceptors in the retinal pigment epithelium ( verhoeff 's membrane ) , while in spectralis it is at bruch membrane [ 11 , 13 , 15 ] . nevertheless , the establishment of different boundaries for total macula thickness calculation can not explain the significant difference between gcipl thicknesses we found in this study . different image resolution , intrinsic reflectance , and analysis algorithms within each software may influence this calculation . we also can not exclude an influence from the different areas analyzed , that is , ellipsoid in cirrus versus the annular in spectralis . however both areas differ only slightly and include the highest density area for ganglion cells . while total macula thickness boundaries can be manually corrected in both cirrus and spectralis , individual layer segmentation correction is possible only in spectralis . we did not observe a significant difference in total macula averages before and after manual correction of inner and outer retinal boundaries in spectralis . this could be explained by the observation that , specifically for the internal limiting membrane and bruch 's membrane , delineation errors occurred mostly in the extreme periphery of the image slice . however , for the gcipl segmentation errors also occur within the 3 mm ring averages . thus gcipl layer segmentation corrections made in spectralis resulted in significantly different values , while values remained significantly higher than with cirrus . in addition , the same difference in thickness measure will impact less on the thicker total macula thickness but more on the thinner gcipl thickness . . a significant correlation between function ( global md ) and morphology ( mt [ 23 , 24 ] or gcipl [ 20 , 25 , 26 ] ) has been demonstrated previously . in agreement , despite the significant difference between cirrus and spectralis measurements , both octs demonstrated a significant positive association with global md . we are not aware of studies directly comparing the diagnostic performance between mt and gcipl in glaucoma . when compared to rnfl , mt had an inferior diagnostic performance in cirrus and in stratus [ 3 , 23 ] . in our study , even though we found no other studies directly comparing auc between mt and gcipl , the auc values found in this study are in accordance with findings from other studies using mt and gcipl [ 7 , 8 , 17 , 20 , 28 ] . though we found a significant difference between oct measurements , the small population analyzed here may limit our findings . also , stage of disease might influence results given that the relationship between structural and functional damage is still not completely understood . including in addition , knowing that age may influence our results , we adjusted all calculations for age . difference in gender distribution is also a concern : cohn et al . did not observe a significant difference between males and females when comparing total db from sap . total macular thickness is about 7.5 m thicker in men than in women . while age and sex differences should be considered when performing disease diagnostics , this is not the main scope of this study as we aimed to analyze measurements from two different octs within the same subject . once this is incorporated , comparison between deviation maps from these octs could contribute to understanding differences between calculations . finally , the spectralis segmentation software used here is a beta version . a definite version , without many changes , the significant difference between measurements from cirrus and spectralis octs does not allow free interchange of machines , for instance , in the follow - up of patients . in a clinical setting , clinicians must be aware that once you change the machine and software analysis , a new baseline for the patient is needed . nevertheless both machines showed similar capability of diagnostic performance in early glaucoma and also in their correlation to functional changes such as standard automated perimetry .

backgroundcd99 is a cell surface transmembrane glycoprotein expressed in various tissues . cd99 is differentially expressed between subpopulations of each tissue and is highly expressed in certain hematopoietic and precursor cells . however , there has been no comprehensive study of cd99 expression in normal skin . we evaluated cd99 expression in normal human skin and developing fetal skin . methodsseventy - five adult skin samples containing normal skin and eight fetal skin samples of different gestational ages were collected . cd99 immunohistochemical staining was performed to evaluate expression pattern in adult and fetal skin samples . cd99 and cd34 expression were compared by double immunofluorescence . resultsin normal adult skin , cd99 was strongly expressed in the membrane of epidermal basal keratinocytes , hair follicle bulges and outer root sheaths , and inner secretory cells of eccrine sweat glands . in fetal skin , cd99 was not expressed on the periderm at 16 weeks of gestation but was expressed in basal cells of fetal skin at around 19 weeks of gestation . cd99 expression became comparable to that of the adult skin after 20 weeks of gestation . cd99 and cd34 were co - expressed in hair follicle outer root sheaths , as seen by double immunofluorescence study . conclusionsthis is the first study examining cd99 expression pattern in normal adult and fetal skin . cd99 tends to be expressed in the basal / precursor cells of epidermis and in hair follicles . these results provide a basis for future investigation on functions of cd99 in the skin and provide a novel potential target for the treatment of dermatologic lesions . purpose . to compare two different spectral - domain optical coherence tomography ( oct ) systems in regard to full macular thickness ( mt ) and ganglion cell layer - inner plexiform layer ( gcipl ) measures and in regard to structure - function correlation when compared to standard automated perimetry ( sap ) . methods . seventeen primary open angle glaucoma patients and 16 controls ( one eye per subject ) were enrolled . mt and gcipl thicknesses were measured by cirrus and spectralis octs . octopus perimeter 101 ( g2 protocol ) reports sensitivity in mean defect ( db ) . differences between measurements were assessed with student 's t - test and bland altman . diagnostic performance was also compared between each parameter calculating the areas under the operator receiver ( roc ) . linear models were used to investigate structure - function association between oct and sap . results . disagreement between octs in both mt and gcipl values was significant . spectralis values were thicker than cirrus . average difference between octs was 21.64 m ( sd 4.5 ) for mt and 9.8 m ( sd 5.4 ) for gcipl ( p < 0.001 ) . patients differed significantly from controls in both octs , in both measurements . mt and gcipl were negatively associated with md ( p < 0.001 ) . conclusions . although oct values were not interchangeable , both machines differentiated patients from controls with statistical significance . structure - function analysis results were comparable , when either oct was compared to sap .

in order to introduce a new plant variety to the markets commercially , it is necessary to register a newly bred variety , which relies upon the results of dus ( distinctness , uniformity , and stability ) tests ; that is , for a new genotype to be registered as a commercial variety , it needs to be distinct ( d ) from all other released varieties , uniform ( u ) , and stable ( s ) for morphological and other evaluated traits [ 1 , 2 ] . therefore , dus test has been established to be the foundation of plant variety protection and also to identify a new variety from reference collection . the new variety should pass legal examinations to be commercialized and receive the certificate for the plant breeder 's rights , a part of which consists of dus tests according to morphological characteristics . the varieties to be assessed are increasing in number where their variability reduces , and the reference collections are expanding because of their internationalization , both of which result in the dramatic increase in expenses associated with these methods . moreover , the existing methods are time consuming , which have altogether led to more necessity for developing a substitutionary , less costly system . thus , the studies on the use of molecular markers in dus testing proving the expected capability of molecular markers have encouraged international union for the protection of new varieties of plants ( upov ) to contemplate the introduction of molecular markers to the dus testing system . nevertheless , before this decision could be made dus testing would benefit from the use as molecular markers that have been shown to be more rapid and cost effective in comparison with morphological traits . in several registration processes such as cultivar identification , primarily , studies were restricted to using the dominant type of markers [ 5 , 6 ] where continuous development of simple sequence repeat ( ssr ) markers has recently resulted in the prevalence of mentioned markers [ 2 , 3 , 7 ] . microsatellite markers have been characterized with multiallelic nature , codominance inheritance , and relative abundance as well as requiring small quantities of dna for amplification which have made these markers efficiently applicable in dus test of rice varieties [ 9 , 10 ] . upov has confirmed the application of ssr markers as one of the commonly practical molecular marker systems for the identification of plant varieties . this marker was previously confirmed to be applied to the distinction between plant varieties as well as complementary features in dus tests where microsatellites were used in dus tests on pepper , canola , and corn . in this study , the efficiencies of ssr markers were evaluated as complementary tools for the distinction of these varieties . in this study , 40 rice varieties consist of 27 varieties created in rice research institute of iran ( rrii ) and 10 local varieties from three regions of guilan , mazandaran , and isfahan in iran , and three international rice research institute ( irri ) varieties were used ( table 1 ) . fifteen seeds of each variety were selected and then 5 g of young and healthy leaves was used for dna extraction . the dna was extracted using the ctab method with minor modifications ( increasing extraction buffer density in two times and replacing mercaptoethanol ( 0.2 percent ) with dithiothreitol ( 30 mm ) ) . twelve pairs of ssr primers ( a pair of primers of each chromosome ) were selected from the panel of 50 from gramene database ( http://gramene.org/markers/microsat/50_ssr.html ) ( table 2 ) . it selected a pair of primers from a mitochondrial dna sequence ( drrcms marker ) ( forward : 5 acctttgggcgatggtt 3 ; reverse : 5 gggtttagagtcgccac 3 ) to detect the impurities in cms line ( ir58025a ) from its cognate isogenic maintainer line ( ir58025b ) which is a prerequisite to obtain pure seeds of hybrid rice as well . pcr reaction was carried out in a total volume of 15 l containing 3 l ( 25 ng ) of template dna , 1 l ( 0.66 mol / l ) primers , 1.5 l 10x pcr buffer , 1.5 l dntps ( 0.2 mmol / l ) , 1.2 l mgcl2 ( two mmol / l ) , 0.2 l taq dna polymerase ( 0.6 u/15 l ) , and 5.6 l ddh2o . an initial denaturation period of five min at 94c was followed by 35 cycles of 60 s at 94c , 30 s at 5666c , 120 s at 72c , and then five min at 72c for final extension . after amplification , the pcr products were separated on 6% ( w / v ) polyacrylamide gel and the frequency of microsatellite polymorphism was calculated based on presence ( 1 ) or absence ( 0 ) of common bands . the genetic similarity between varieties was calculated using the dice coefficient , and a dendrogram showing the genetic relationship of the 40 varieties was constructed using the unweighted pair group method with the arithmetic mean ( upgma ) features of the ntsyspc v2.02 statistical analysis package : ( 1)gdnl=2n112n11+n10+n01 . accordingly , n11 is the number of bands ( alleles ) in both individuals ; n00 is the number of bands ( alleles ) absent in both individuals ; n10 is the number of bands ( alleles ) in i , n01 is the number of bands ( alleles ) present in j , and n is the total number of all bands ( alleles ) . effective number of alleles ( ae ) in each ssr locus was calculated by the following formula : ( 2)ae=1pi2 , where pi is the frequency of ith allele for each locus . the polymorphic information content ( pic ) of microsatellite loci was calculated according to the following formula : ( 3)pic=1j=1npij2 , where pij is the frequency of the jth allele for ith marker and the summation extends over n alleles . moreover , the discriminative power of molecular markers ( dj ) and the best combination of microsatellite markers ( xk ) were estimated using the following steps and formulas . there are n(n 1)/2 different pairs in a set of n individuals . based on this , ci is the ith pattern of the given jth primer in which formula is ( 4)ci = pinpi1n1 . moreover , for the jth primer , cj is equivalent to the summation of the different ci for all i patterns generated by the primer : ( 5)cj=i=1ici=i=1ipinpi1n1 . therefore , the discriminating power of the jth primer and the best combination of k primers are equal to ( 6)dj1cj=1i=1ipinpi1n1,xknn12j=1kcj . the 12 microsatellite primers used for this study generated totally 83 polymorphic fragments with an average of 6.91 alleles per locus . among these markers , rm316 with 13 alleles and rm55 with three alleles had the highest and lowest variation , respectively . effective number of alleles was calculated from 2.44 ( rm161 ) to 9.52 ( rm316 ) with an average of 4.90 per locus . additionally , the pic was estimated from 0.52 ( rm161 ) to 0.9 ( rm316 ) with an average of 0.74 per locus . the discriminating power ( dj ) ranged between 0.57 ( rm161 ) and 0.93 ( rm316 ) with an average of 0.78 per locus ( table 2 ) . the most of microsatellite markers had a high pic and discriminating power . however , a few markers had a low range of pic and discriminating power such as rm55 and rm161 . our results agree partially with those of hashemi et al . , who utilized 10 microsatellite markers to characterize the genetic diversity in a group of 16 iranian rice hybrids . pic is regarded as one of the important features of the molecular markers and can be used to evaluate the differentiation ability of the markers . the range of similarity among varieties was from 0 to 1 with an average of 0.26 and variance of 0.063 for all microsatellite markers . similarity values in between varieties were 0 for 66 pairs of varieties ( supplementary file 1 ; see supplementary material available online at http://dx.doi.org/10.1155/2015/965073 ) and similarity value had been 1 just for one pair of varieties ( ir58025a versus ir58025b ) . ir58025a is a cms line , and , for detecting the cms line from its cognate , isogenic maintainer line ( ir58025b ) was used ; the polymorphism of a mitochondrial dna sequence ( drrcms marker ) between some of cms population and their fertile lines is verified in both lines ( figure 1 ) . dendrogram resulted from cluster analysis using upgma algorithm based on the dice similarity coefficient and could discriminate all varieties from each other except for two isonuclear lines ( ir58025a and ir58025b ) ( figure 2 ) . as a result , total microsatellite markers could detect most of the improved varieties ( group a ) from local varieties ( group b ) . however , sang - e - jo and hassan - saraie as local varieties stood with improved varieties in group a , because sang - e - jo and sepid - rood were used as recipient parents for ghaem-1 variety . as can be observed in the dendrogram , sang - e - jo , sepid - rood , and ghaem-1 have been in the same subcluster . these varieties are shafagh and kadous that improved from two irri lines as ir67015 - 94 - 2 - 3 and ir64669 - 153 - 2 - 3 , respectively . sazandegi , purified from lenjan local mass , and shiroudi improved by cross between deylamani as a local variety and khazar as an improved variety . consequently , jahesh stood with other varieties obtained from tarom such as tarom - jolodar , sang - e - tarom , and tarom - milad in group b , and sazandegi with lenjan and shiroudi with deylamani constructed identical subcluster in group b. there is an interesting result in the third group ( group c ) , in which three varieties , namely , tabesh , pouya , and parto , constructed the same subcluster with tarom - mahali . these varieties are mutant of tarom - mahali . although the rice varieties in this study were from different rice breeding programs in iran , microsatellite markers correctly grouped them depending on their respective group , local or improved varieties , in the dendrogram . , who analyzed 43 rice samples using microsatellite markers and obtained a similar classification of varieties according to their breeding programs . in addition , it obtained results from cluster analysis of each marker which have shown the ability of some of the markers as unique identification key for some of the varieties ( table 3 ) . for finding the best combination of markers that result in the obtained result of using all markers in discrimination of all varieties , first , markers were chosen one after another in a way to minimize xk , that is , the number of pairs of distinct varieties for each primary compound in each step . in the first step , rm316 was chosen which distinguished the highest pairs of varieties from each other among n(n 1)/2 pairs of varieties and made the amount of dj maximum . in the second step , the compound of each n 1 remaining marker with the chosen marker of previous step was tested in order to determine the most efficient compound that minimizes the amount of xk . in this step , compounding the rm271 with previous marker left the lower number of variety pairs undetermined . in subsequent steps , finally , adding rm154 to previous markers could decrease expected number of undetermined pairs of varieties calculated from 39.27 to 0.03 which should practically reach one pair of variety . there were 41 pairs of varieties nondistinct from each other in calculating similarity coefficient among varieties and then cluster analysis of varieties using rm316 that had the highest discriminating power ( 0.93 ) among markers . by adding consequent markers in accordance to distinguish the ability to this marker , the number of undistinguished pairs of varieties was decreased to one ( table 4 ) . reliable identification of similar varieties is so difficult in plant species by morphological characteristics alone , because morphological and physiological characteristics are limited [ 25 , 26 ] . accordingly , using molecular markers as additional information is inevitable in registering plant varieties considering their benefits . dna markers can be utilized to simply and rapidly detect varieties or approve the distinctiveness of a varietal impostor . for identification and characterization of rice varieties and the testing of hybrid rice lines , using sts and ssr markers was significantly easier than using typical grow - out tests that included growing plants to maturity and evaluating purity based on morphological characteristics [ 28 , 29 ] . also , microsatellite markers have been utilized in the previous same study to distinguish traditional rice ( oryza sativa l. ) varieties from each other in cuba . in this research , the results showed that microsatellite markers easily could be used for identification of rice varieties . sequence - defined biopolymers , such as proteins and nucleic acids , incorporate backbone and side - chain constituents that endow these macromolecules with the ability to fold into well - defined secondary and tertiary structures . the complexity and functionality of these folded biopolymers has spurred intensive research toward a predictive understanding of the relationships between their sequences , structures , and functions . computational strategies to engineer proteins and nucleic acids have matured to the point where de novo design of elaborate new protein and nucleic acid structures can be conducted with some reliability . the development of computational tools for the design of proteins also provides a valuable blueprint for building analogous tools to enable design of other abiotic folded oligomeric systems , termed foldamers . a key component of many protein design programs is the discretization of side - chain degrees of freedom by representing side chains as conformational isomers , termed we describe general methods to build rotamer libraries for peptidomimetic foldamers , and apply these methods specifically to a family of peptoid foldamers composed of n - substituted glycine monomers . in this way , we aim to remove a fundamental roadblock and enable design methods for diverse foldamers incorporating abiotic monomer types . foldamers are a class of oligomeric molecules for which noncovalent interactions dictate the self - organization of stable secondary and tertiary structures . there is now a veritable bestiary of foldamer species and hybrids . a large community of researchers are actively developing functional peptoids to address a diverse set of goals . the intensity and diversity of these efforts punctuates the need for general computational tools to aid in the pursuit of many complex design tasks . in this study , we provide the computational tools and develop the theoretical background necessary to design the next generation of folded and functional peptoid oligomers . the solid phase submonomer peptoid synthesis protocol introduced by zuckermann et al . facilitates the introduction of a myriad of side - chain types by utilization of readily available primary amines as synthons . peptoids have been the subject of considerable research aimed at developing sequence structure as well as structure function relationships . these efforts have established that peptoids can populate a range of secondary structure types and that peptoids exhibit strong interactions between side chain and backbone degrees of freedom . despite the absence of stabilizing backbone hydrogen - bonds , peptoids have been shown to fold , and their folds can be predictably controlled by variation of the monomer sequence to achive a desired functionality . additionally , new native chemical ligation strategies have been developed to ligate peptoids to peptides . this ligation protocol will enable the synthesis of hybrid biomacromolecules aimed at achieving advanced functions . rotamer libraries are an essential part of the protein design toolbox . based upon the observation that protein side chains populate distinct areas of side - chain dihedral angle conformation space and that dihedral angle conformations are in some cases strongly dependent on the adjacent and backbone dihedral angles , side - chain conformations have been grouped together into bins that represent frequent rotational - isomers and given the moniker rotamer . for canonical amino acids ( caas ) , methods to find these regular clusters of conformations rely on statistical analysis of the protein data bank ( pdb ) . the large number of structures for each side chain across the spectrum of allowed backbone dihedral angles allows for the determination of relative rotamer energies by fitting a boltzmann distribution to the population of side - chain conformations . current rotamer libraries are backbone dependent : given and backbone torsion angles , the rotamer library specifies a set of allowable rotamers , the estimated probability of each rotamer , and some measure of the deviation within the cluster of similar conformations represented by that rotamer . enumerating the set of side - chain conformations and their likelihoods allows for rapid searching of low energy side - chain conformation in discrete steps . these libraries are key constituents in several structural bioinformatics approaches including methods for placing side chains on homology models , protein structure prediction , and protein design . statistically derived rotamer libraries are not feasible for systems with few experimental structures . in the case of peptoids , there are fewer than 20 experimentally determined high - resolution peptoid structures , the largest of which includes only 16 residues . this limitation necessitates the use of mm based force - field energy calculations and qm calculations to derive estimates for the relative conformer energies of peptoid foldamers . from these computed estimates , we can then establish a rotamer - like treatment of side - chain probabilities . previous attempts at developing rotamer libraries for noncanonical -amino acid and -amino acid side chains have also used mm - based force - fields . there are many conceivable algorithmic options to determine rotamer minima placement . in this study , we devised two distinct rotamer library development methods and find they achieve similar levels of quality in structure prediction and design task performance . we evaluated the quality of these rotamer libraries within the context of the molecular modeling suite rosetta . in addition to creating rotamer libraries for peptoids , we introduce several substantial modifications to the rosetta code to enable design with noncanonical backbone chemistry . rosetta was initially built to predict protein function , but has been expanded to include protocols for docking , protein design , rna structure prediction , and other macromolecular structure and design tasks . the code is widely distributed and used by more than 1500 research groups worldwide . by incorporating our work on peptidomimetic design into rosetta , we make available a large number of computational protocols for scoring , kinematics ( moving the backbone ) , docking , and optimization . this enables utilization of peptoids in existing rosetta design protocols , such as designing peptoid sequences to interact with proteins . below we describe the methods used for the creation of rotamer libraries , methods used to evaluate the assumptions made in utilizing rotamers , as well as methods used to characterize the performance of the resultant rotamer libraries . we have recently compiled a database of all high - resolution peptoid structures ( the peptoid data bank ) . we additionally included structures of peptoid / peptide hybrids bound to sh3 and ww domains . selection criteria and modifications to structures are detailed in the supporting information . in the set of oligo - peptoid structures , there are 9 different peptoid side chains , at 87 positions and with 93 unique conformations . four of the most commonly observed peptoid side chains ( nspe , 21 ; nmeo , 17 ; nphe , 15 ; ns1ne , 11 ) , making up 69% of unique conformations in the peptoid data bank , are described in the main body of the text and are shown in scheme 1 . side chains for which fewer experimental examples exist ( nary , 16 ; nrch , 5 ; npe , 4 ; n1nap , 3 ; nrpe , 1 ) are detailed in the supporting information . in addition to the 9 side chains mentioned above , we chose 45 side chains to include within the rosetta framework , based on their prevalence in the literature and the ease with which they could be incorporated into oligomers via standard synthetic routes . a full list of the 54 peptoid side chains that were incorporated into rosetta and for which we created rotamer libraries are shown in figures s1s7 . previous studies of oligo - peptoid structures have defined both atom names and dihedral angles in different ways . our choice of atom names was strongly influenced by peptide atom naming for compatibility with protein modeling programs and is similar to that used by huang and co - workers . whereas previous studies have defined the 1 dihedral angle relative to the preceding carbonyl carbon , rosetta requires that the atoms that define a dihedral angle be contained within the residue unit . we therefore deviated from past work by defining 1 with respect to the c1 atom ( c in peptides ) . our atom name and torsion angle naming conventions are shown for an nspe residue in scheme 2 . atom names shown as red italics in torsion angle definitions are atoms in the preceding ( ) or following ( ) residues in a oligo - peptoid chain and not shown in scheme . in our analysis of peptoid side chain conformations below , we use the rotamer notation originally set forth by lovell et al . for its clarity and brevity , where p is used to represent plus ( trans , 180 ) . for side chains with dihedral angles that are commonly found at angles other than 60 , 60 , or 180 , the angle is shown in the notation . when referring to the 1 rotamer wells , unless otherwise noted , we use m and p to refer to 90 and 90 , respectively ( e.g. , a side chain listed with a rotamer of mm would have a 1 dihedral angle near 90 and a 2 dihedral angle near 60 , while an m90 rotamer would have a 1 dihedral near 90 and a 2 dihedral near 90 ) . qm energy landscapes of 1 , n , and c as a function of the preceding- dihedral in the context of a tertiary amide bond ( tab ) model ( scheme 3 ) were computed . the tab model contains the minimum set of atoms needed to simultaneously describe the preceding- and 1 dihedral angle of an oligo - peptoid residue . all qm geometry optimizations and single point energy calculations of tab models were evaluated at the b3lyp/6 - 311+g(d , p ) level of theory using gaussian09 . atoms are named as in scheme 2 , and names shown as green italics represent atoms from preceding residues in a oligo - peptoid chain . two sets of tab models were constructed that combinatorially sample the dihedral angle and the 1 dihedral angle when the dihedral is in either a cis or trans conformation . the set of cis- input tab models sampled from 40 to 40 in 10 intervals . the set of trans- input tab models sampled from 140 to 140 in 10 intervals . both sets of tab model inputs sample the 1 dihedral angle through a full rotation in 10 intervals . two additional sets of tab model inputs were constructed to quantify the changes in the location of the energy minima . these additional sets sample the same cis and trans ranges but set 1 to 90 or 90. both sets of inputs were subjected to qm geometry optimization where only the dihedral angle values were fixed followed by a single point energy calculation . the calculations of the versus c and versus n energy landscapes are detailed in the supporting information . the results are shown in figures 2 and s8 . to compare the qm based energy landscape to the rosetta mm_std(34 ) ( a mm - based force - field ) based energy landscape , we calculated the energy landscapes for four of the most common side chains in the context of both backbone - independent ( bbi ) and backbone - dependent ( bbd ) model compounds ( scheme 4 ) . the bbi model was used to quantify the dependence of side - chain energy landscapes . models were initialized at 10 intervals for each angle , keeping and the other dihedral angles fixed . all other parameters were allowed to optimize . to further quantify the concerted effects that , , and might have on the energetic landscape , we repeated the dihedral scan on the bbd model system while keeping the , , and angles fixed at 0 , 90 , and 180 , respectively , as a majority of peptoid structures have these approximate backbone dihedral configurations . all qm geometry optimizations and single point energies were evaluated at the b3lyp/6 - 311+g(d , p ) level of theory using gaussian09 . similar energy scans were repeated with the same model compounds using the rosetta mm_std force - field without geometrical optimization . we have previously shown that using the mm - based mm_std energy function in rosetta , we can produce rotamer libraries for proteins and noncanonical -amino acids that are comparable to statistically derived rotamer libraries . to explore and compare the relative merits of context and scoring approximations we describe two methods : ( 1 ) quantum mechanically seeded ( qms ) : which uses a highly accurate qm based scoring function , but a minimal model of the peptoid backbone / environment , and ( 2 ) k - means clustering ( kmc ) : which uses a more efficient molecular mechanics scoring function , but more extensively explores a model of the peptoid backbone . both protocols require a residuetype parameter file that instructs rosetta how the side chain is allowed to move and how the energy of the residue is to be calculated . drew et al . the residuetype parameter file describes the atom names and types , the chemical connectivity of the side chain and an ideal internal - coordinate representation used when rosetta needs to create new instances of the side chain . a qm geometry optimization of a bbd dipeptoid , using gaussian09 at the b3lyp/6 - 311+g(d , p ) level of theory , is used to generate the idealized internal coordinates that serve as a starting point for the kmc and qms protocols . workflow for the k - means clustering ( kmc ) and quantum mechanically seeded ( qms ) rotamer library construction protocols . boxes shaded in green are qm geometry optimizations of backbone - dependent ( bbd ) or backbone - independent ( bbi ) models ; red , inputs to rosetta ; blue , geometry optimization using the rosetta mm_std energy function ; yellow , identification of local energy minima . our previously described rotamer library construction protocol was adapted for peptoids as shown in figure 1 . model system , a peptoid residue with an acetylated n - terminus and a n - dimethylamide c - terminus , has been previously used to examine backbone - side - chain interactions in peptoids as it mimics the environment of the side chain with its own backbone and the backbones of the preceding and following residues . the additional dependence of peptoid side chain conformations on the preceding- backbone dihedral angle necessitated modification to the protocol to sample and produce rotamer libraries that are dependent on the preceding- angle as well as and dihedrals . the and backbone dihedrals were sampled through a complete rotation in 10 intervals to produce 36 and 36 bins . in the set of rosetta compatible peptoid data bank structures , cis and trans preceding- backbone dihedrals were sampled between 30 to 30 and 150 to 150 in 10 intervals to produce 14 bins . a combinatorial sampling of all ,, bins yields 18 144 backbone bins . for each backbone bin , dipeptoids were constructed that combinatorially sample the side - chain dihedral angles . side - chain dihedral angles were sampled at user - defined sets of angles relating to the number of angles , the chemical connectivity and the expected number of rotamers ; typically 10 intervals ( e.g. , nspe has 36 1 and 36 2 samples to produce 1296 dipeptoids for each ,, bin ) . the set of minimized dipeptoids was then k - means clustered based on the similarity of the minimized side - chain dihedral angles . the final rotamers for a given backbone dihedral bin are the side - chain dihedral angles of the lowest energy dipeptoid from each cluster in that bin . the side - chain conformation of each final rotamer was sampled about the local minima until the energy increased by 0.5 rosetta energy units ( reu ) to obtain an approximation of the width of the local energy minima . this local energy minima width is used as a proxy for the standard deviation of the side - chain conformations in a single rotamer bin . we devised an alternative methodology to use qm energy scans of a bbi model of the peptoid side chains as shown in figure 1 . the minimum energy wells identified from qm energy landscapes were used as starting points for lower level mm optimization and energy evaluation on a bbd model system . the bbi model containing each side chain is initialized in gaussian09 into discrete intervals spanning the entire 1n ( where n is the total number of angles ) with a fixed backbone dihedral angle at either 0 for cis or 180 for trans conformations and allowed to geometrically minimize while keeping the dihedral fixed . the qm derived minima from those single point energy scans converge to small clusters . from each cluster , this set of angles for both cis and trans conformations serves as the complete set of bbi energy wells . the bbi energy well dihedral angle coordinates were then initialized onto the bbd model in rosetta in the same range of backbone bins as the kmc protocol . the bbi energy well coordinates were then minimized using the rosetta mm_std scoring functions and their relative energies were used to determine rotamer probabilities assuming a boltzmann distribution of the resulting energies . all geometry optimizations of the molecules in the bbi qm - derived minima scans were performed at the b3lyp/6 - 311+g(d , p ) level of theory . rotamer recovery benchmarks tested the performance of the two rotamer libraries at reproducing the low energy packed side - chain conformations observed in experimental structures in both the presence ( when applicable ) and absence of the symmetry related partner molecules in the oligo - peptoid structures . a rosetta protocol was written to carry out fixed - backbone side - chain repacking using the packrotamersmover followed by a comparison between the original and repacked side - chain conformations . the packrotamersmover simultaneously repacks all side - chain positions using a metropolis monte carlo simulated annealing procedure that attempts to find the lowest energy set of side - chain conformations given the current backbone conformation . calculations were performed with the ex1 , ex2 , and ex3 command line flags set to true ; and the extrachi_cutoff flag set to 0 . these flags force rosetta to sample 1 , 2 , and 3 rotamers at their mean one standard deviation at all positions . in order to model only side - chain conformations described by the rotamer library , the use_input_sc flag is set to false to exclude the experimentally determined side - chain conformation of the input structures in sampling . a residue s side chain is considered predicted correctly if the angle of the repacked model was within 20 of the position in the native structure . although peptoid oligomers can readily be synthesized to incorporate a wide diversity of side chains , there are only a handful of experimentally determined peptoid structures . there has yet to be a systematic analysis of side - chain conformations in all peptoid structures that explores side - chain conformations given all energetically feasible backbone conformations . additionally , to determine if a rotameric treatment of peptoid side chains is appropriate , it is necessary to establish that the dihedral angle values of the experimental side - chain conformations cluster and that there is a multidimensional interdependence between the locations and frequency of those clusters . here we briefly describe our results for a small set of side chains for which there are sufficient experimental data to allow for proper validation . following these case studies , first , we explore our ability to fit existing structures by quantifying the distance between experimental side - chain conformations and the corresponding closest rotamers in our library . second , we evaluate the performance of our rotamer library in the context of a peptoid design task by evaluating repacking of peptoid side chains within existing peptoid crystal structures . lastly , we evaluate our ability to model peptoid side chain conformation at an experimentally validated protein - peptoid interface ( in comparison to x - ray crystal structures of the interfaces ) . in all three cases , we achieved good performance for rotamers developed from both the quantum mechanically seeded ( qms ) and k - means clustering ( kmc ) methods . this strongly indicates that peptoid side chain conformations can be approximated by a rotameric treatment and that our rotamer libraries are suitable for several design tasks . peptoids , unlike peptides , have greater flexibility around , with some monomer types readily populating both cis and trans ( e / z ) angle conformations with a substantial range of deviation around the ideal angles of 0 and 180 for cis and trans , respectively . the rotation of the preceding- torsion angle has been both predicted and observed to alter the preferred 1 value , as well as pyramidalization of the backbone nitrogen ( n ) and backbone carbonyl carbon ( c ) atoms . to quantify the effect has on these side - chain conformations , and to justify development of a backbone dependent rotamer library that includes this additional dihedral angle variability , we explored the -dependent energy landscapes . in the development of rotamers based upon the tab ( scheme 3 ) , system we sought to ensure that rotamers developed using an inflexible amide bond model would be applicable for design in systems with slight deviations from ideal bond lengths , angles , and dihedral parameters . the rotamer libraries used by rosetta only include information about side - chain torsion angles with respect to the backbone torsion angle values . in order to use a rotameric treatment of peptoid side - chain conformations , the degrees of freedom that are not described in the rotamer library need to be relatively stable or invariant to perturbations of the backbone torsion angles . to verify that this was the case for peptoids , we quantified the relationships between the backbone dihedral angle and the 1 dihedral angle ( figure 2 ) as well as carbon ( c ) and nitrogen ( n ) dunitz amide bond puckering parameters ( figure s8 ) . from these energy landscapes , there are several notable dihedral angle dependencies . we found that the 1 rotamer energetic preferences for the tab model were most significant ( 0.5 kcal / mol ) at extreme dihedral angle deviations from planarity . the 1 dihedral angle minima as denoted by the solid and dashed lines ( figure 2 ) had notable ( 20 ) deviations from 90 and 90 , dependent upon the dihedral angle . this result confirms the necessity to develop rotamer libraries dependent on not only the and , but also on backbone dihedral angles , as the effects of deviations on 1 have now been quantified and have been found to be significant . the n and c dunitz parameters varied as a linear function of . these puckering trends are built into the 1 energy landscapes , resulting in the observation that even with extreme dihedral angles and deviation of the amide bond from planarity , the 1 energetic preferences vary by only 0.5 kcal / mol . this stability buttresses our confidence that idealized amide bonds are a reasonable starting approximation for -dependent peptoid rotamer libraries . from this result , we are confident that rotamer libraries that include an dependence will be able to accurately capture the energetic preferences of diverse peptoid oligomer species . energy landscapes were generated by fixing the dihedral angles of the tab model to simultaneously achieve the desired 1 or . cis- angles can be found on the left , trans- can be found at the right . crystal structure data are shown as circles and crosses for angles and parameters found in cyclic and linear peptoid structures , respectively . in the trans- energy landscapes , crystal structure values for the nary monomer are not plotted , as this parameter does not match the chemistry of the tab model system . the minimum energy parameters are plotted across the full range of values as well as for positive ( solid line ) and negative ( dashed line ) 1 values . all molecules were minimized and energies evaluated at the b3lyp/6 - 311+g(d , p ) level of theory , and heatmaps generated using the lowest energy for each plot as the zero kcal / mol baseline . we used two protocols to generate backbone dependent ( bbd ) rotamer libraries for peptoid side chains in order to enable comparisons between approaches for identifying rotamers , scoring conformations , and modeling backbone - side - chain conformation interdependence . the first protocol is a modification of the method previously published to calculate -amino acid side - chain rotamer libraries , referred to as the kmc method . it uses the molecular mechanics ( mm ) based rosetta mm_std energy function in the context of a bbd molecule to evaluate rotamer energies . previous studies using quantum mechanics ( qm ) have shown a complex interaction between the peptoid side chain and backbone . while qm is accurate , it is also computationally intensive and can not solely be used to create a full rotamer library or for side - chain repacking and design calculations . our second method is a new rotamer library creation protocol that uses input from qm calculations carried out on backbone - independent ( bbi ) molecules and is referred to as the qms method . the qms method then passes these qm - bbi minima to rosetta to estimate interactions with the backbone . thus , each method uses a different strategy to reduce the complexity of the problem and arrive at a protocol with computational efficiency sufficient to allow calculation of rotamer libraries for multiple side chains . there are more than 200 peptoid side chains that are synthetically feasible ; however , only 9 different side chains have been used in experimentally determined peptoid structures . it is therefore essential that both protocols are general methods that do not incorporate or require structural information from experimentally determined peptoid structures as training data . we tested these two methods to determine if our rotamers , in conjunction with the mm based rosetta mm_std energy function , could accurately capture the behavior of peptoid side chains to the extent required by tools developed for protein modeling . we show that the two methods , despite taking different approaches , ultimately find similar rotamers . a discussion of the rotameric states of the side - chain conformations observed in the current set of peptoid structures and how they compare to the side - chain conformations of the rotamer libraries produced here are included in the supporting information . additionally , an excerpt of the side - chain dihedral angles from the rotamer library are included in supporting information tables s1s4 . the energy landscape generated in the initial steps of the qms protocol uses the bbi model ( scheme 4 ) . this minimal model was chosen due to the fact that qms single point energy evaluation of the energy landscape including the backbone and torsions for all possible dihedral angles is computationally intractable . the reduced representation contains all of the side - chain atoms but only the torsion angle of the preceding residue . to investigate if this model is sufficient , we computed side - chain dihedral ( 1 and 2 ) energy landscapes of four common side chains with bbd dipeptoid models at common low energy backbone conformations using qm and the rosetta mm_std energy function and compared them to the landscape of bbi models ( figure 3 ) . most notable is the absence of the 1 minima near 90 that are present in the bbi model energy landscape but absent in the bbd energy landscape for the four common side chains . the absence is the result of steric interaction between the c1 of the side chain and the c atom of the backbone which has a fixed dihedral of 90. if the angles are fixed at 90 , we observe an absence of 1 minima near 90. these results can be rationalized by the similar repulsive effects observed in the syn - pentane model system . the loss of the 90 minima seen in the backbone - dependent energy landscapes is analogous to the repulsive effects observed in syn - pentane which has been extrapolated to explain forbidden rotamers at certain and dihedral angles of amino acids . additionally , the appearance of the m0 and p0 rotamers of nspe and nphe in the bbd molecule in peptoid structures indicates that the bbi screen is not always successful in capturing the complete ensemble of side - chain conformations observed in peptoid structures . the qms protocol is also limited to two angles , as a complete screen of side chains with many rotatable bonds is computationally intensive and often intractable with qm . differences between cis and trans bbi models show that while the relative energy varies , the location of the minima remain similar between the two ( figure s9 ) . the minima from the bbi model landscape found as small clustered circles in figure s9 are represented as large diamonds in the bbi portion of figure 3 . these minima are then initiated onto a bbd model and the model is allowed to minimize using linear gradient minimization and the rosetta mm_std scoring function . the diamonds on the bbd portion of figure 3 represent these minimized rotamer positions in the presence of the backbone model . it can also be observed that the qm and mm bbd energy landscapes closely resemble one another , with only minor differences . rosetta and other computational protein modeling packages use side - chain dihedral angles in rotamer libraries to discretize the search for low energy side - chain conformations ( protein repacking ) or sequences ( protein design ) for a given backbone conformation . an accurate rotamer library will contain side - chain dihedral angle values close to values observed in experimentally determined structures . rotamer libraries should be succinct for computational efficiency , but also sufficiently comprehensive to enable sampling of a large fraction of energetically accessible conformations . to test the completeness of the rotamer libraries produced by the kmc and qms rotamer library creation protocols , we carried out rotamer library coverage tests . these tests calculate the rmsd ( in degrees ) between the experimentally observed side - chain rotamer conformation and the closest rotamer in the given rotamer library . results of the rotamer library coverage tests for the four frequently observed side chains are shown in figure 4 and table 1 . results of the other experimental side chains are shown in figures s10s14 and table s5 . for comparison , these tests were additionally carried out for phenylalanine and methionine side chains in protein structures in the top 8000 data set using the dunbrack 2002 bbd rotamer library , tables 1 and s7 . fixed backbone rotamer energy landscapes for nphe , nspe , ns1ne , and n-(propyl)-glycine side chains in a backbone - independent ( bbi ) and backbone - dependent ( bbd ) context . crystal structure dihedral angle values are shown as circles and crosses for those observed in cyclic and linear peptoid structures , respectively . x - ray crystal dihedral angles for the different side chains are plotted only for the monomers in which the backbone dihedral angles were observed to be within 20 of the fixed backbone dihedral angles used in the energy landscape calculations . the minima from the bbi model landscapes ( figure s9 ) are represented as large diamonds in the bbi portion of the figure on the left . the diamonds on the right portion of the figure represent these rotamer positions in the context of the bbd model after rosetta mm_std energy function minimization . all landscapes underneath a qm header had energies evaluated at the b3lyp/6 - 311+g(d , p ) level of theory . heatmaps were generated using the lowest energy as each plot s zero kcal / mol and zero reu ( rosetta energy units ) baseline for qm and rosetta , respectively . for each of the four most frequent peptoid side chains , either the kmc or qms rotamer libraries contained rotamers with angles that are , on average , within less than 20 of experimental side chain values . for the nspe side chain , only the p90 and p0 rotamers are experimentally observed and accurately modeled by rotamers created with both the kmc and qms method . the experimental points occupy a wide energy valley that spans from a 2 of 90 to 30. the kmc method performs better than the qms as it is able to find a low probability rotamer in this valley while the qms method predicts p0 rotamers closer to a 2 of 30. for the nmeo side chain , the experimental conformations adopt the traditional m , p , and t positions . the 3 dihedral angle values did not form tight clusters in the kmc protocol ( data not shown ) . this results in a relatively large rmsd value ( table 1 ) despite 1 and 2 values being accurately predicted ( figure 4c ) . for the nphe side chains , the rmsd value for the kmc rotamers is just over the 20 threshold while the qms is significantly higher at 38. the bbi screen of the qms method misses the high energy m0 and p0 rotamers ( figure 3a ) , and the qms rotamer library does not include these conformations . the qm energy landscape with the backbone present shows an elongated energy valley for 2 ( figure 3a ) . the five experimental examples with 2 near 0 are missed by the qms method and contribute to the high rmsd in table 1 . the three experimental points near the m0 rotamer are also significantly different from the values in the kmc library . deviations in 1 can arise from pyramidalization which can greatly affect the positioning of the atoms making up the 1 dihedral angle , potentially influencing the 1 calculated value . when the experimental points with a 2 near 0 are omitted , the qms rotamers have almost the same rmsd to the experimental values as the kmc rotamers for m90 and p90 rotamers ( 18.25 and 18.71 , respectively ) . for the ns1ne side chain , both the kmc and qms protocols perform as well as the dunbrack 2002 library for protein data . the large steric bulk of the naphthyl group interacting with the c2 atom of the ns1ne side chain and peptoid backbone restricts the allowed conformations of the side chain . only the pp rotamer is observed in the experimental data set , and both methods predict this rotamer accurately . with few exceptions , rotamers observed in peptoid structures are found in the rotamer libraries produced by both methods . both the kmc and qms protocols produce similar rotamers with similar dihedral angles . the kmc protocol suitably evaluates longer side chains and is able to find side - chain conformations that involve backbone interactions such as the nphe rotamers with 2 near 0. the dunbrack 2002 rotamer library for the 20 canonical peptide amino acids performs better than our rotamer libraries perform on peptoid side chains . however , compared to the top 8000 data set , there are far fewer examples of peptoid structures than protein . additionally , the data set employed for the protein comparison is heavily pruned to only include the highest quality structures available ; an option we do not have for peptoids . we investigated if the combination of the peptoid rotamer libraries and the mm_std scoring function have sufficient discriminatory power to recapitulate the side - chain conformations in the experimentally determined structures . we therefore undertook side - chain conformation recovery benchmarks similar to those employed in the early development of protein design methodologies . all non - hydrogen atoms in the monomer were used to calculate the rmsd . positions from the top 8000 data set with less than eight neighbors ; two residues are considered neighbors if their neighbor atoms ( c for glycine , c for all others ) are within 10 of each other . rotamer library coverage plot for nphe , ns1ne , nmeo , and nspe peptoid side chains . interpolated torsions and standard deviations of the closest rotamer in the rotamer library based on the backbone dihedral angles of each experimental point are shown as crosses , where the center of the cross is at the mean and the length represents 1 standard deviation . rotamers for the k - means clustering ( kmc ) method are shown as red crosses and quantum mechanically seeded ( qms ) method are shown in blue . each example in the set of rosetta compatible peptoid structures was repacked with rosetta using rotamer libraries from the kmc and qms protocols and in the presence ( where applicable ) or absence of crystal contacts from symmetry related partners . the predicted side - chain dihedral angles of the repacked structures were compared to those in the experimental structure . a angle is judged to be correctly predicted ( recovered ) if it is within 20 of the experimental value . results of the rotamer recovery benchmark in structures containing only peptoid residues are shown in table 2 and figure 5 . rotamer recovery rates in proteins improve with additional context about the environment the side chain is in . for surface positions , that additional context can be provided by the atoms from neighboring chains in the symmetery related cyrstalographic neighbors . the surrounding atoms in a protein s core also provide additional context and can help determine correct rotamer position . previous studies on proteins found that rosetta achieved an over all rotamer recovery of 75% for 1 and 53% for 1 + 2 using the mm_std energy function and the dunbrack 2002 rotamer library . a recovery of 59% for 1 and 37% for 1 + 2 was achieved for surface positions , and a recovery of 91% for 1 and 71% for 1 + 2 was achieved for core positions using the same energy function and rotamer library . the increased recovery of protein cores strongly suggests that , for a given position , the influence of surrounding side chains can enhance the discretization of low energy side - chain conformations . in the currently available set of peptoid structures , the number of neighbors a given side chain has is more comparable to the surfaces rather than cores of proteins . both the kmc and qms rotamer libraries are able to correctly predict the 1 conformations of more than 60% of peptoid positions both in the absence and presence of crystal structure contacts ( table 2 ) . the kmc and qms rotamer libraries achieve rates of peptoid side - chain recovery comparable to the recovery rate of the dunbrack 2002 library for protein side chains at surface positions . percent rotamer recovery absent crystal contacts totals have been adjusted to account for structures determined by nmr . our ability to recover correct rotamers is dependent on the quality of the rotamer library coverage . the 12ac1 - 9-c structure , an nspe 9-mer , has the highest rate of side chains recovered ( figure 5 ) because the rotamers produced by the kmc and qms protocols have a low averaged rmsd compared to the experimental side chain conformations for nspe ( table 1 ) . in contrast , the 12ab4 - 16-m structure contains only nmeo and nary side chains and has the lowest fraction of rotamers recovered in the set . the rotamer library coverage for the nmeo and nary side chains is more complete relative to nspe ( tables 1 and s5 ) , and the effect is that we poorly predict the side - chain conformations within the 12ab4 - 16-m structure . to get a better understanding of how rosetta will behave in repacking a peptoid side chain in the core of a globular protein or buried at protein protein interfaces , we carried out rotamer recovery benchmarks in the context of the neighboring peptoid molecules in the solid state defined by the crystallographic symmetry transformations ( table 2 ) . the addition of crystallographic partners has been shown to increase the rate of rotamer recovery at protein surface positions . although not a perfect analogue of a protein s hydrophobic core , increasing the number of neighboring residues through the addition of crystal contacts reduces the number of conformations assessable to the given peptoid position . additionally , crystal contacts can direct the side chain into conformations that are lower in energy as a result of the additional contacts . this allows rosetta to choose a rotamer closer to those observed in the crystal structures . for example , nspe-4 and nspe-8 monomers in the peptoid data bank structure 12ac1 - 9-c are both correctly predicted by both the kmc and qms rotamer libraries . however , the angles of the selected conformations are closer to the values in the experimental structure when crystal contacts are included in rotamer repacking . this effect is highlighted in figure 5 with the side - chain conformation predicted with crystal partners ( red ) closer to the experimental conformation ( gray ) than without the crystal information ( blue ) . there are currently too few structures to determine if crystal contacts direct side chains into off - rotamer conformations . however , for the two available nmr solution structures , rosetta has a 1/2 recovery of 67%/75% for kmc and 80%/75% for qms . our ability to accurately model peptoid side chain conformations with a rotameric treatment in rotamer recovery benchmarks supports the notion that peptoids are indeed rotameric . like peptides , each peptoid side chain is rotameric at varying levels as a result of complex side chain to backbone intraresidue and steric interactions . overall , we find that the currently available set of experimental side - chain conformations are sufficiently modeled with our predicted rotamer conformations . to more thoroughly evaluate the degree to which peptoids are rotameric will likely require additional peptoid structures with greater numbers of side - chain - side - chain contacts , tighter packing , and a more diverse palate of side chains . however , with the currently available set of rosetta - compatible peptoid data bank structures , we find that experimental side - chain conformations cluster well and that those clusters correspond to minima found in qm and rosetta mm_std energy landscapes . it is clear that some rotamers will simply not be observed due to steric clashes with the backbone ; other predicted rotamers have not been observed due to the small size of the current database of peptoid structures . this initial study indicates that similar to peptides , peptoids also exhibit rotamer preferences . furthermore , this finding suggests that protein modeling tools can be readily adapted to accommodate them . nguyen and co - workers have deposited three structures of sh3 domains bound to inhibitory peptides in which each peptide has a single proline position mutated to a different peptoid side chain . these three structures provide us an opportunity to test our ability to recover native rotamers in hybrid design contexts . structure 1b07 contains two chains ( labeled a and c in the deposited structure ) , while 2sem and 3sem contain four chains each , two pairs of protein / peptide interactions ( a / c and b / d , respectively ) . as with the rotamer recovery of the oligo - peptoid structures , each structure here was repacked and the side - chain dihedral angles were compared to those in the experimental structure . results of the rotamer recovery benchmark in structures of peptoid / peptide hybrids are shown in table s9 and figure s15 . rosetta is able to recover the rotamers of the 1b07 and 2sem structures using both the kmc and qms rotamer libraries . we are not able to recover the rotamer of the peptoid side chain from the 3sem structure . rosetta places the side chain in an alternative conformation ( data not shown ) . the peptoid side chain in the 3sem structure branches at the second side chain atom and makes few contacts in the crystal structure . additionally , the average b - factors of the atoms in the 3sem side chains are greater than 40 , indicating uncertainty in the exact position of the peptoid side chain in this experimental structure ( table s9 ) . for these reasons we exhibit good performance for both structures with well - resolved peptoid side chains , but recognize that the small sample size prevents us from generalizing further . we present a general method for creating rotamer libraries needed for rational design of peptidomimetic oligomer structures . we apply this method to the peptoid backbone and show performance is comparable to protein side - chain rotamer libraries derived from statistical analysis of the protein data bank ( pdb ) for protein surface positions . this pipeline relies on mm and qm simulations in lieu of statistical analysis because far fewer crystal structures of peptoids exist than for proteins . given the reliance on physics - based methods , we expect this method can be applied to several other diverse peptidomimetic scaffolds ( such as -peptides , d - amino acid , and hybrid oligomeric systems ) . a notable advantage of this method is that it can be used to build rotamer libraries for any specified side chain . this is demonstrated by the development of rotamers for over 50 peptoid side chains ( shown in figures s1s7 ) that are capable of being incorporated via standard peptoid synthesis protocols . peptoid data bank structure 12ac1 - 9-c and side - chain conformations after being repacked with ( a ) kmc rotamer libraries or ( b ) the qms rotamer libraries . experimental side - chain conformations are shown in gray , repacked side chains in blue , and repacked in the context of the symmetry related crystal partners in red . positions for which the same rotamer was chosen in both contexts are shown in purple . comparisons between qm evaluations of peptoid side - chain conformations and our rotamers show good agreement between the qms and kmc rotamer library construction pipelines . our comparisons with qm suggest we capture key features of the side - chain conformational landscape . we also find agreement with the side - chain conformations observed in x - ray crystal and nmr structures of peptoid oligomers ( figure 4 ) . there are currently too few experimentally determined peptoid structures to derive peptoid rotamer libraries by statistical analysis . the currently available peptoid structures are of small oligomers ( < 20 residues ) and are dominated by crystal contacts and local structure interactions ( comparable to protein surface positions ) . we show that our rotamers agree with experimental side - chain conformations with rmsd values comparable to best - in - class protein rotamer libraries for -amino acids at surface positions ( figure 4 and table 1 ) . a large number of modifications to the rosetta design framework were required to enable peptoid design with these rotamer libraries ( described in the supporting information ) ; most notably , allowing for the use of bbd rotamer libraries that include preceding- in addition to and . these modifications to the rosetta design procedure allowed us to evaluate our performance on repacking tasks , and again we find that these rotamer libraries will be sufficient for peptoids and hybrid peptoid - protein design tasks ( such as designing peptoids to interrupt protein protein interfaces ) . despite the rosetta energy function being optimized for biological molecules in aqueous media , it performs surprisingly well at reproducing the side - chain conformations of relatively short oligo - peptoid structures in nonaqueous media . this indicates that the side - chain conformations of peptoids are primarily determined through local interactions . adding peptoid design capabilities to rosetta allows access to kinematics , optimization , and scoring methods that enable a vast array of design and modeling tasks for peptoid , peptoid these rotamer library development methods are also extendable to other noncanonical backbones and peptidomimetic scaffolds . future work will include adding capabilities to model and design several other peptidomimetic oligomer scaffolds . a key remaining challenge not addressed here , is to model mixed oligomeric systems such as oligomers containing -amino acids and peptoids . additional study is required to determine if rotamer libraries derived individually for peptoid and -amino acids will perform well in these hybrid settings . in cases where a peptide side chain precedes a peptoid side chain , both side chains would be separated by only two bonds along the backbone and we speculate that this mixed system would require rotamer libraries specific to the joint between the two oligomeric systems . for cases in which peptoid side chains are n - terminal to peptide , the proximal side chains will be separated by four bonds and it is likely that the rotamers derived in this study would perform well in this mixed setting . other key areas for future work include the need for developing better methods to estimate the unfolded state energies ( sometimes referred to as the reference energy ) as well as new methods for dealing with larger side chains . as we design , build , and refine peptoids , we will increase the diversity and number of structures and thus increase our ability to score and judge peptoid designs . we thus intend to bootstrap our way toward design capabilities for both peptoid and mixed protein peptidomimetic systems that approach pure protein design in accuracy and breadth of application .

identification and registration of new rice varieties are very important to be free from environmental effects and using molecular markers that are more reliable . the objectives of this study were , first , the identification and distinction of 40 rice varieties consisting of local varieties of iran , improved varieties , and irri varieties using pic , and discriminating power , second , cluster analysis based on dice similarity coefficient and upgma algorithm , and , third , determining the ability of microsatellite markers to separate varieties utilizing the best combination of markers . for this research , 12 microsatellite markers were used . in total , 83 polymorphic alleles ( 6.91 alleles per locus ) were found . in addition , the variation of pic was calculated from 0.52 to 0.9 . the results of cluster analysis showed the complete discrimination of varieties from each other except for ir58025a and ir58025b . moreover , cluster analysis could detect the most of the improved varieties from local varieties . based on the best combination of markers analysis , five pair primers together have shown the same results of all markers for detection among all varieties . considering the results of this research , we can propose that microsatellite markers can be used as a complementary tool for morphological characteristics in dus tests . peptoids are a family of synthetic oligomers composed of n - substituted glycine units . along with other foldamer systems , peptoid oligomer sequences can be predictably designed to form a variety of stable secondary structures . it is not yet evident if foldamer design can be extended to reliably create tertiary structure features that mimic more complex biomolecular folds and functions . computational modeling and prediction of peptoid conformations will likely play a critical role in enabling complex biomimetic designs . we introduce a computational approach to provide accurate conformational and energetic parameters for peptoid side chains needed for successful modeling and design . we find that peptoids can be described by a rotamer treatment , similar to that established for proteins , in which the peptoid side chains display rotational isomerism to populate discrete regions of the conformational landscape . because of the insufficient number of solved peptoid structures , we have calculated the relative energies of side - chain conformational states to provide a backbone - dependent ( bbd ) rotamer library for a set of 54 different peptoid side chains . we evaluated two rotamer library development methods that employ quantum mechanics ( qm ) and/or molecular mechanics ( mm ) energy calculations to identify side - chain rotamers . we show by comparison to experimental peptoid structures that both methods provide an accurate prediction of peptoid side chain placements in folded peptoid oligomers and at protein interfaces . we have incorporated our peptoid rotamer libraries into rosetta , a molecular design package previously validated in the context of protein design and structure prediction .

malaria remains a serious global health problem , killing more than one million people per year . the number of malaria cases has fallen by more than 50% in 43 countries over the past decade . a modeling analysis of malaria prevention activities in 34 african countries suggested that about 730,000 lives were saved between 2000 and 2010 , with nearly three quarters of those since 2006 . funding commitments for malaria have increased nearly 15-fold , from approximately us$ 100 million in 2003 to nearly us$ 1.6 billion in 2010 ; interest and commitment at global and country levels are very high . however , the problem of malaria parasite transmission remains enormously grave in sub - saharan africa where at least 85 to 90% of deaths are attributable to the disease [ 47 ] . malaria transmission is driven by a complex interaction of the vector , parasite , human host , and the environment , and is governed by different ecological and social determinants [ 8 , 9 ] . globalization and urbanization with their inherent developmental activities and associated ecological transformations have a significant impact on malaria epidemiology [ 10 , 11 ] and have invariably exacerbated the situation . malaria transmission depends markedly on local environmental conditions and other compounding factors , that is , presence of drug - resistant parasites and insecticide resistant vectors [ 12 , 13 ] , environmental changes , economically driven human population increase and migration , poverty levels , climatic changes , natural disasters and political upheavals , adaptability of malaria vectors to changing environments [ 17 , 18 ] and limited investment in research , drug discovery , and optimisation of malaria vector control programmes . transmission patterns and severity of malaria are influenced by the geographic attributes and the socioeconomic environment that vary significantly by city , season , and age group . accordingly , entomological profiles and clinical patterns are known to vary between urban , periurban , and rural environments . well - developed urban areas are mostly fringed by underdeveloped and inadequately serviced periurban areas experiencing the highest population growth rates and often lacking infrastructure . malaria transmission in peri - urban areas is mostly ascribed to increased vector breeding created by the agricultural and construction activities , lack of drainage of surface water [ 18 , 22 , 23 ] , human vector contact due to poor housing and overcrowding , and low immunity in children under five and pregnant women , thus increasing the risk of severe disease . in zambia , between 1950 and early 1980s , vector control reduced malaria cases to a notifiable disease in most urban areas . ngandu and coworkers reported the resurgence of malaria cases in urban and peri - urban lusaka . in vivo sensitivity tests were also conducted with plasmodium falciparum patients in lusaka , but whether these infections were acquired in urban lusaka itself or in rural areas was not clear . owing to malaria cases resurgence and paucity of entomological data , specific local investigations to appraise and confirm malaria transmission in peri - urban lusaka were required before approaches to malaria vector control could be considered . we report here on malaria vectors , parasite prevalence rates in febrile patients and knowledge and attitudes of the community pertaining to malaria , precedent to the implementation of the integrated vector management ( ivm ) strategy . zambia is a landlocked country in southern africa with an estimated population of 13 million people , 45% are children below 15 years of age . malaria is endemic across the entire country with transmission peaks coinciding with the rainy season from november to april . this study was conducted in two spatially segregated and randomly selected peri - urban locations of lusaka district ; chazanga and kalikiliki ( figure 1 ) during the cold - dry season from may to july 2003 . the two sites have similar ecological characteristics and stretch out in an epidemiological zone characterized by low malaria transmission . mosquito larvae were collected from breeding sites using who - standard 250 ml dippers , transported to the insectary at the national malaria control centre in lusaka , and reared to adults while being fed on 1 part yeast and 2 parts dog biscuit . adults were maintained on 10% sugar solution at 25 2 centigrade temperature and 7080% relative humidity . mosquito breeding sites were characterized into three different categories : transient , semipermanent , and permanent . transient breeding site refers to temporal water collections , semi - permanent ones are those that would persist for a considerable period of time . adult mosquitoes were collected by the pyrethrum spray catch ( psc ) between 06:00 hrs and 08:00 hrs in randomly selected households . anopheles mosquitoes were identified morphologically using standard keys for anophelines of southern africa [ 29 , 30 ] and to species by the polymerase chain reaction ( pcr ) molecular method of scott et al . . plasmodium falciparum infection was determined among febrile patients at health facilities in the study sites . blood from randomly selected subjects who presented to the health center with febrile symptoms and consenting to participate was screened for parasite species and gametocytes by microscopy using 4% giemsa thick and thin blood smears for 30 minutes . the age range of subjects was stratified into three age categories : 6 months < 5 , 5 to < 15 , and 15 years and over . participants with positive slide tests were offered free treatment with artemisinin - based combination therapy ( act ) according to zambia national malaria control programme treatment policy guidelines . a pretested structured questionnaire was administered to 150 randomly selected respondents , tested for malaria , to determine community knowledge and attitudes as regards malaria , family demographic data , and possibility of malaria importation from rural areas . data was collected and entered in excel spread sheets ( microsoft corporation ) and statistically analyzed by employing epi info version 3.2.2 . the chi - square ( ) test was used to determine the differences in parasite prevalence between age categories . ethical approval for the research a freely administered informed consent was given to respondents and householders for participation in the study . of 1840 larvae collected , 66% ( 95% ci : 65.768.1 ) were from transient ( gardens and abandoned building foundations ) , 28% ( 95% ci : 25.629.6 ) semipermanent ( abandoned shallow wells and ditches that followed in the wake excavations for building sand or quarrying ) and 6% ( 95% ci : 5.47.7 ) permanent water bodies ( perennial streams and dams ) ( figure 2 ) . the density of anopheles larvae was comparatively higher in semipermanent ( 31.7% ) followed by the permanent ( 25% ) and transient habitats ( 17.5% ) ( figure 2 and table 1 ) . anophelines constituted 12.83% ( 95% ci : 8.717.9 ) of the 203 adult mosquitoes collected ( table 1 ) . the mosquito male - to - female ratios and densities per room was 0.59 to 0.26 and 1.7 to 15 for anopheles and culex , respectively . all specimens from kalikiliki ( n = 11 ) and chazanga ( n = 7 ) , amplified for an . gambiae ss . and only 1 from chazanga amplified for an . arabiensis ( figures 3 and 4 ) . a total of 297 randomly selected febrile patients were recruited into the study ( table 2 ) . seventy - six ( 25.6% ) were positive for malaria parasites with 100% plasmodium falciparum parasite monoinfection . among the positive slides , 75 ( 98.7% ) exhibited ring form trophozoites and only 1 ( 1.3% ) showed gametocytaemia . the parasitemia in febrile patients per age group was 31.8% ( 95% ci : 23.242.2 ) for the 04 years group , 25.7% ( 95% ci : 13.541.3 ) for 5 to 15 years , and 23.3% ( 95% ci : 17.429.6 ) for the 15 years and over ( p > 0.05 ) . of the 150 respondents 18% ( 95% ci : 12.424.6 ) were male and 82% ( 95% ci : 75.487.3 ) were female . sixty - two per cent showed awareness of what to do when they suspected malaria and only forty - six per cent were knowledgeable about vector control interventions . family demographic data showed an average of seven residents with at least one child under five years per household . there was positive association between knowledge and malaria prevalence in peri - urban lusaka ( p < 0.05 ) . funestus [ 34 , 35 ] , with great divergence in their malaria transmission potential , spatial segregation , and temporal heterogeneity [ 36 , 37 ] . the pioneering malaria control efforts in the country [ 38 , 39 ] stimulated unprecedented enthuse in entomological studies [ 36 , 4044 ] . rivulorum although their role in malaria transmission in zambia is yet to be established . urban areas are perceived not to support significant levels of malaria transmission . in this study , three kinds of mosquito breeding habitats : transient , semipermanent , and permanent were characterized with appreciable spatial heterogeneity ( figure 2 ) . gambiae is known to exploit small open temporal habitats with less predation , increased warmth , and more algae . however , more anopheles larvae were collected from semipermanent habitats than from permanent and transient habitats ( figure 2 and table 1 ) . this could explain the role of urban development related activities in supporting high malaria transmission levels as observed in peri - urban lusaka . while formal urban development typically reduces mosquito densities , informal urbanization has been shown to alter the vector species composition within the an . gambiae complex in sub - saharan africa , . to illustrate , earlier studies conducted in zambia indicated 100% an . nevertheless , the profound demographic and extensive environmental changes that have followed in the wake of urbanization have changed the stratification of the vectors . gambiae ss . validates the premise that informal urban development does transform vector species composition . arabiensis , a species that is typically difficulty to control by irs and itns , and the predominance of the an . gambiae ss . which is characteristically amenable to control by irs and itns could have implications for the malaria control programme . the sympatric - existence of these vectors demonstrates the need for an integrated approach for malaria vector control . this study was characterized by low number of mosquito collections due to the unfavorable prevailing environmental conditions during the cold season , lack of data on chromosomal forms of an . transmission determining parameters , that is , vector infectivity . however , early entomological work in zambia reported a sporozoite rate of 1.4% in an . notably , there is still a clear paucity of data on malaria vector bionomics in the country . malaria had been known to be hyperendemic in hot riverine valleys with perennial transmission , meso - to hypoendemic on plateaus , and hypo - endemic in urban areas of zambia . between 1969 and 2000 , parasite rates ranged from 2.0 to 26.4% across the country , with parasite species of 86.8% p. falciparum and 13.2% p. malariae in ndola rural . by 1999 , parasite species was over 97% p. falciparum . these findings are corroborated in this study with 25.3% parasitaemia among febrile patients with 100% p. falciparum monoinfections . this upsurge of frequency of febrile malaria was further aggravated by the development of chloroquine resistance . deployment of effective control tools has transformed the epidemiological profile from countrywide high endemicity to three distinct epidemiological strata : very low transmission and parasite prevalence of < 1% , low transmission ( < 10% ) , and persistent high transmission ( > 20% ) . the prevalence rate of malaria in children under five years is dependent on the intensity of transmission and declines with age as immunity develops and is thus a good indicator of a recent transmission of malaria . the highest prevalence of malaria in zambia occurs in this age group across the country . in this study , frequency of febrile malaria was highest ( 31.8% ) in the 04 years age group and lowest ( 23.3% ) in the 15 years and above group . there was no significant difference in parasitaemia in febrile patients of the three age categories ( p > 0.05 ) suggesting a nonimmune population and an area of low transmission . the above 10% parasitaemia observed in children under 5 years of age confirmed that malaria had again become endemic in peri - urban lusaka . the knowledge and attitudes survey indicated the need for intensified information , education and communication ( iec ) on malaria and its prevention . the 46% knowledge level on vector control interventions indicated a weakness in individual efforts to prevent the disease . population expansion and its health impact has been epitomized by sub - saharan africa . in many malaria endemic countries , including zambia , the population has doubled in the past two decades , thus greatly increasing the absolute numbers of those at risk . this was demonstrated in peri - urban lusaka where family demographic data showed an average of seven residents with at least one child less than five years per household . thus , suggesting that congestion in households was probably one of the factors contributing to the increased transmission of malaria in these settings . it has equally been established that human migration contributes markedly to malaria transmission . in areas of endemicity , encroaching transmission has been demonstrated in areas previously free of transmission and local transmission has been conclusively demonstrated in many african cities [ 55 , 56 ] . these findings are corroborated in this study which confirmed local transmission in lusaka as 80% subjects with definitively diagnosed malaria had no history of travel . it was established that there is no significant contribution of migration towards malaria transmission in peri - urban lusaka ( p > 0.05 ) . local transmission of malaria was further strongly inferred by high parasitaemia in children under the age of five and the presence of gametocyte bearers and efficient vectors in the community that perpetuated the transmission cycle . congestion in households together with the appreciably low levels of knowledge on control and prevention compounded the situation . the pragmatic data reported on here was an essential prerequisite of evidence - based and effective vector control efforts . the high malaria infection rates in peri - urban lusaka could be ascribed to the definitively demonstrated local transmission . gambiae complex species and characterization of their breeding attributes required an integrated vector management ( ivm ) approach to effectively control transmission . it is noteworthy , that this preintervention study had limitations as the surveys were conducted during the dry season which markedly influenced the malaria vector and parasite populations . clearly , the malaria epidemiology in peri - urban lusaka required an integrated approach involving irs and itns against the adults and larval source management ( lsm ) against the aquatic stages . information education and communication ( iec ) to increase awareness and knowledge about malaria vector control needed to be intensified . following this study , ivm was introduced in lusaka with irs and itns as main thrust interventions and iec has been strengthened . this has reduced malaria parasite rates to appreciably minimal levels ( < 1% ) . to clear the residual transmission , lsm is being implemented in lusaka . while monitoring and evaluation of vector control interventions has been strengthened , it is imperative that a comprehensive entomological and epidemiological surveillance system is established to detect any increase in the malaria case load . hydrocephalus refers to the accumulation of excessive cerebrospinal fluid ( csf ) within the brain or cranial vault . medical treatment of hydrocephalus , which aims to reduce csf production , is usually only palliative . surgical shunting of csf from the ventricular system to another body cavity , usually the peritoneum and less often the right atrium , is the gold standard treatment in people , and can provide a superior long - term prognosis . ventriculoperitoneal shunt ( vps ) placement is the most commonly utilised surgical procedure in human and veterinary patients with hydrocephalus . despite this , complications following vps surgery are common , with failure rates in people of up to 50% within the first year . mechanical failure remains the most common cause of vps malfunction in people , with shunt obstruction being the leading cause ( 46% ) , followed by shunt disconnection , infection ( with reported rates of 410% and 7.6% ) and over - drainage . recent veterinary studies have reported complication rates of 22% , 25% and 29% . in one study , shunt occlusion and infection occurred in 11% and 8.5% of animals , respectively , although the patient numbers were vastly lower than those in human studies . retrograde migration of the peritoneal catheter ( pc ) to the ventricle , the subgaleal space and the subcutaneous tissues of the head , neck and chest , often with coiling of the catheter in areas of loose skin , has been described in several human case reports , although this remains a much less common mechanical complication . martinez - lage et al reported an incidence of 0.6% in 500 human shunt procedures , and sayers reported pc migration resulting in shunt malfunctions in only 3/1390 cases . there is very limited literature regarding postoperative complications of vps in veterinary patients and a distinct lack of information regarding the incidence of retrograde migration of the pc , especially in cats . to our knowledge only a single case report exists in the japanese veterinary literature , describing pc migration to the subcutaneous tissue of the dorsal chest in a cat and drainage tube obstruction 5 years postoperatively in another cat , along with a further report describing multiple episodes of kinking of the pc in a cat . this case report describes the retrograde migration and subcutaneous coiling of the pc of a vps , detected 72 h following percutaneous aspiration of a vps - associated seroma in a cat with non - communicating , unilateral hydrocephalus . a male neutered domestic shorthair cat , aged 4 years and 10 months , was presented with a 3 week history of episodic , anticlockwise circling . neurological examination revealed mild generalised ataxia and proprioceptive deficits , a wide - based pelvic limb stance , bilaterally absent menace responses , poor visual tracking and good visual placing responses . t2-weighted hyperintense material , which was suppressed on fluid - attenuated inversion recovery images , filled the uniformly enlarged ventricle . a moderate - to - severe mass effect was evident , with contralateral deviation of the midline and compression of the thalamus , the rostral colliculus and the rostral aspect of the mesencephalon , indicating caudal subtentorial herniation and raised intracranial pressure . there was a reduction in thickness of the cerebral mantle overlying the enlarged ventricle and asymmetry of the calvarium . there was rostral bulging of the frontal bone with subsequent reduction in the volume of the right frontal sinus , within which an incidental small frontal sinus cyst was identified ( figure 1 ) . mris of the brain demonstrating right unilateral congenital hydrocephalus : ( a ) axial t2-weighted , ( b ) axial t2 fluid - attenuated inversion recovery and ( c ) dorsal three - dimensional fast imaging employing steady - state acquisition the mri findings were compatible with non - communicating hydrocephalus , with gross changes indicative of chronicity , although no underlying aetiology could be identified . levetiracetam ( 25 mg / kg iv q12h , keppra ; ucb ) and prednisolone ( 1 mg / kg po q24h for 2 weeks followed by 0.5 mg / kg q24h , prednidale ; dechra ) were initiated , with minimal response after 2 months . a pc ( open end with wall slits , standard , pliant , barium impregnated , 90 cm [ reference 43551 ; medtronic ] ) was placed intraperitoneally via a right lateral laparotomy and anchored to the abdominal wall with a chinese finger - trap nylon suture . the pc was subcutaneously tunnelled cranially in a straight line to the level of c1c2 on the dorsal midline . using a right lateral rostrotentorial approach , a semi - lunar incision was made over the right calvarium from the caudal aspect of the frontal sinus to the level of c1c2 . two holes were drilled in the skull approximately 30 mm and 25 mm cranial to the occiput and 1.4 mm lateral to the midline . the dura was incised and the cerebral cortex perforated with a rivulet ventricular catheter ( barium - impregnated , 15 cm [ reference 41701 ; medtronic ] ) via the first hole , which was secured into the right ventricle with two nylon sutures anchored to the second hole . the ventricular catheter was connected to a csf flow control valve ( fcv ) ( ultra - small , low - low pressure [ reference 22017 b - ll ; medtronic ] ) at the level of c1c2 , which was secured to the pc caudally . the pc was concertinaed into three loops ( perpendicular to the axis of the neck ) before connection to the fcv , to allow for movement . satisfactory positioning of the vps was confirmed on postoperative ct scan ( figure 2 ) . recovery was unremarkable and postoperative neurological examination revealed mild generalised ataxia and proprioceptive deficits , bilateral mydriasis and reduced menace responses with good vision . postoperative analgesia was provided with methadone ( 0.10.2 mg / kg iv q46h , comfortan ; dechra veterinary products ) and subsequently with buprenorphine ( 0.02 mg / kg iv q68 h , vetergesic ; ceva animal health ) . the patient was discharged 48 h postoperatively with gabapentin ( 10 mg / kg po q12h for 1 week ) , amoxicillin / clavulanic acid ( 12.5 mg / kg po q12h for 2 weeks , synulox ; zoetis uk ) , levetiracetam ( 25 mg / kg po q12h for 2 months , then q24h for 1 month ) and prednisolone ( 1 mg / kg po q24h for 1 week , then 0.5 mg / kg q24h for 3 weeks , then 0.5 mg / kg q48h for 6 weeks ) . there was mild deterioration of the neurological examination 12 h postoperatively ; bilaterally , mildly reduced nasal sensation and mydriasis had developed , both of which had resolved at 36 h and 2 weeks postoperatively , respectively . ct rendering sagittal image demonstrating satisfactory positioning of the ventriculoperitoneal shunt a 48 h period of loose faeces was reported 2 weeks postoperatively , which resolved spontaneously . increased vocalisation and polyphagia was reported until at least 8 weeks postoperatively , likely secondarily to prednisolone . neurological examination had improved 13 weeks postoperatively ; a very mild generalised ataxia and proprioceptive deficits and bilaterally reduced menace responses were detected . persistent , generalised pruritus was reported ; a flea infestation had been identified and treated with selamectin ( stronghold ; zoetis uk ) ; however , no environmental parasiticide treatment had been applied . a soft , non - painful , 4 cm diameter subcutaneous swelling was present at the level of the csf fcv , which had developed over the previous 3 weeks . ultrasound performed prior to seroma aspiration revealed a small length of coiled vps tubing within the seroma ( consistent with original placement ) and thus radiography was not performed . serous fluid ( 15 ml ) was aspirated using a 5 ml syringe and 23 g needle . in - house cytology ( cytospin ) revealed a mixed - cell population consisting of non - degenerate neutrophils and macrophages , and no evidence of intra- or extracellular bacteria . palpation post - aspiration did not reveal an abnormal volume of coiled catheter within the seroma . meloxicam ( 0.05 mg / kg po q24h , metacam ; boehringer ingelheim ) was instituted along with cold packing of the region q8h . the swelling reformed after 3 days but was palpably firm and approximately 7 cm in diameter . radiography confirmed cranial migration and subcutaneous coiling of the entire pc within the subcutaneous tissues at the level of the fcv ( figure 3 ) . lateral radiograph demonstrating proximal migration and coiling of the entire peritoneal catheter within a seroma at the level of c1 the swelling was surgically explored under general anaesthesia . no adhesions between the subcutaneous tissues and the catheter tubing were noted . the pc was flushed to confirm patency and re - tunnelled to the right abdominal wall and secured intraperitoneally . at the time of writing , the owners reported a persistent , marked improvement in coordination and gait , and cessation of circling and compulsive pacing since vps placement . to our knowledge , this is the second report of migration of the pc of a vps in a cat . in people , pc migration mostly occurs within the first 3 months of shunt placement , as occurred in this case . multiple episodes of under - shunting due to kinking of the pc , requiring invasive care , were reported in a cat within the first 3 months of surgery , although subcutaneous coiling of the pc was not reported in that case . no shunt complications were seen 360 days postoperatively compared with 24 months in the current case . the current treatment recommendation is surgical removal and replacement of the pc within a new subcutaneous tunnel . ultrasonographical evaluation of the seroma in this case confirmed a normal length of pc at the level of the fcv and thus radiography to confirm the intra - abdominal positioning of the pc was not performed prior to seroma aspiration . consequently , it is unclear whether pc migration began prior to or following seroma aspiration . if the former is true , then perhaps this afforded increased mobility of the fcv and contributed to seroma formation . conversely , the size of the fcv represents a significant foreign body in a cat and thus seroma formation would have been feasible in the absence of pc migration . pang and wilberger suggested that subgaleal fluid re - absorption may create negative pressure , drawing the pc proximally . alternatively , upward migration may be afforded by a combination of suction from negative intraventricular pressure created due to csf drainage , pushing from positive intra - abdominal pressure ( iap ) secondarily to physiological processes or excessive csf volume within the abdomen , a tortuous subcutaneous tunnel and a lack of appropriate fixation of the proximal and distal ends of the vps tubing . in the current case , the subcutaneous tunnel was created in a straight line using minimal dissection and the pc was secured in the abdomen by a nylon chinese finger - trap suture , ensuring adequate fixation . soft faeces were documented for 48 h within the initial postoperative period , although the occurrence of significant tenesmus could not be confirmed . the owners also reported a noticeable and persistent ( for at least 8 weeks postoperatively ) increase in vocalisation , seemingly associated with polyphagia , which was thought to be related to tapering prednisolone therapy . it is feasible that raised iap generated by increased vocalisation and possibly tenesmus may have facilitated pc migration prior to seroma aspiration . windlass effect , whereby a proximal anchoring point , such as granulation tissue , allows tension from repeated flexion extension movements of the head , noted in some hydrocephalic infants , to be transmitted to the pc , creating a proximal winching effect . no obvious adhesions anchoring the pc to the surrounding tissues ( which might have created a however , considering the range of motion of the feline head and neck it is possible that a similar windlass effect may be generated by the repetitive flexion , extension and twisting movements that occur during self - grooming and scratching , potentially increasing the risk of pc migration in cats . a high frequency of self - grooming and pruritus was observed in this case prior to representation and seroma aspiration , likely secondary to a flea infestation , which may have contributed to pc migration and also possibly to seroma formation due to increased movement of the fcv . windlass effect to cause pc migration in an infant with compulsive flexion extension head movements . they subsequently re - tunnelled the pc away from the axis of the head movements . further studies would be required to establish whether an optimal shunt pathway exists in cats that would significantly reduce the risk of pc migration . it is possible that the seroma itself may have caused irritation and increased self - grooming , exacerbating the risk of pc migration . however , the owners reported a generalised pruritus which was not specifically at the level of the seroma and there was no reaction to seroma palpation , making this less likely . a facilitatory role of loose subcutaneous tissue has been suggested , whereby reduced resistance to catheter movement augments the proximal migration and coiling process . several human reports describe the coiling of a migrated catheter within subcutaneous regions with a previous history of fluid accumulation . softer , more mobile skin may further explain the higher incidence of catheter migration to the proximal subcutaneous regions in children than in adults . the seroma in the current case created an area of loose subcutaneous tissue , which may have aided pc migration and coiling . furthermore , compared with human infants , cats have more substantial amounts of loose , mobile skin , especially along the dorsolateral occipital , cervical and cranial thoracic regions , which may further predispose this species to proximal pc migration . minimal undermining of the subcutaneous tissues when creating the tract for the shunt tubing may be even more crucial in cats than in people . martinez - lage et al proposed a mechanism whereby rapid release of the pc from its anchoring point around the abdominal scar could be facilitated by abrupt stretching . subsequent leakage of csf from the catheter and distension of the subcutaneous tunnel could reduce resistance to retrograde movement of the catheter . the distension of the subcutaneous tract could act as a buffer and account for the initial lack of neurological signs of raised intracranial pressure seen in some people with vps migration , as was seen in this case . normal feline activities ( eg , jumping , climbing and hunting ) could trigger the above proposed mechanism , perhaps creating an increased risk of pc migration in cats that are younger , more active and/or have an outdoor lifestyle compared with those cats that are older , less active and/or confined indoors . seroma aspiration may have exerted negative pressure on the pc , drawing it proximally and facilitating either dislocation of the catheter from the peritoneum , or proximal migration of an already displaced , extraperitoneal catheter , or perhaps both . gentle suction was applied using a small - volume ( 5 ml ) syringe to minimise negative pressure . however , considering that ultrasound identified a normal volume of catheter within the seroma prior to aspiration , that the entire pc was radiographically confirmed to be coiled within the seroma within 3 days of aspiration and that no further complications occurred following revision surgery , seroma aspiration may have been a significant contributor to pc migration and subcutaneous coiling in this case . dominguez et al considered that the memory of the shunting device , due to coiled packaging , could enable recoiling within the subgaleal space . however , a similar case of upward catheter migration and subgaleal coiling has been described with uncoiled catheter packaging . furthermore , head motion of this infant was prevented , discrediting the windlass mechanism in this case . in addition , no marked dissection or subgaleal fluid was documented at the revision surgery , making reduced resistance to tube movement and negative suction pressure less likely mechanisms , respectively . no association was found between the type of shunting device employed and the tendency towards upward migration . the main limitations of this case report are the lack of imaging of the distal pc to confirm its location prior to seroma aspiration and the lack of veterinary literature documenting vps catheter migration in cats to assess the significance of the observations made . migration of the pc of a vps most commonly occurs within the first 3 months following placement , with several possible mechanisms of migration suggested . preventive measures include proper fixation of the proximal and distal ends of the vps , minimal dissection when creating the subcutaneous tunnel and straight placement of the tubing . shunt positioning away from the axis of repetitive head movements may also be beneficial , although the optimal shunt pathway in the cat is yet to be determined . in the current case , an increased frequency of vocalisation ( possibly associated with polyphagia secondary to prednisolone therapy ) and diarrhoea causing subsequent raised iap and repetitive self - grooming / scratching creating a windlass effect may have contributed to catheter migration . thus , cats displaying a high frequency of vocalisation , tenesmus and/or self - grooming , including long - haired cats and those receiving treatment ( eg , prednisolone ) , or with comorbidities that may exacerbate these behaviours , may be at a higher risk of vps shunt migration . measures to reduce the frequency of postoperative self - grooming and/or pruritus ( eg , encouraging owners to groom their cat , reducing stress , minimising the duration of steroid treatment and preventing / treating comorbidities ) , appropriate prevention and/or prompt treatment of conditions predisposing to raised iap and moderate exercise restriction , particularly within the first 3 months , may help reduce the risk of peritoneal catheter migration in feline patients following vps placement . negative suction pressure exerted by aspiration of a fcv - associated seroma may have initiated or contributed to pc migration and subcutaneous coiling in the current case . subsequently , diagnostic imaging should be offered , ideally pre- and postaspiration of a vps - associated seroma , with appropriate aspiration technique and equipment employed ( applying slow , controlled suction using a small - volume syringe with or without a butterfly catheter ) to minimise negative suction pressure and the potential risk of catheter migration .

globalization and urbanization with their inherent developmental activities and ecological transformations impact on malaria epidemiology . entomological factors involved in malaria transmission in periurban lusaka were assessed prior to vector control reintroduction . data was collected through standard entomological and epidemiological protocols and a pretested structured questionnaire . larval habitats were characterized as transient ( 43% ) , semipermanent ( 36% ) , and permanent ( 21% ) . anopheles arabiensis and an . gambiae ss . were the only vectors identified . a shift in vector population was noted , with the later outnumbering the former . plasmodium falciparum monoinfection rates were 25.6% ( 95% ci : 20.930.7 ) ( n = 297 ) . parasitaemia was 31.8% ( 95% ci : 23.242.2 ) , 25.7% ( 95% ci : 13.541.3 ) , and 23.3% ( 95% ci : 17.429.6 ) in under 5 , 5 to 14 , and above 15 age groups , respectively . low knowledge levels on vector control tools with an average of 7 residents per household were also observed . this study confirmed a local malaria transmission paradigm . the epidemiology necessitated deployment of an integrated vector management strategy with intensified information education and communication . case summary ventriculoperitoneal shunt placement is the most commonly utilised surgical treatment for hydrocephalus in human and veterinary patients . migration of the peritoneal catheter is an uncommon but well - documented complication in people , usually occurring within the first 3 months postoperatively , although only a single feline case report exists . a ventriculoperitoneal shunt was placed in a domestic shorthair cat , aged 4 years and 10 months , following a diagnosis , with mri , of unilateral , non - communicating hydrocephalus . diarrhoea , increased vocalisation and pruritus were reported within the first 3 months postoperatively . a shunt - associated seroma developed , which was aspirated under ultrasound guidance . within 3 days , the entire peritoneal catheter was subcutaneously coiled at the level of the seroma . the peritoneal catheter was replaced within the abdomen via a new subcutaneous tunnel . no further complications had occurred 24 months following revision surgery.relevance and novel information this is the second report describing peritoneal catheter migration in a cat . repetitive head and neck movements during self - grooming , raised intra - abdominal pressure secondary to vocalisation and tenesmus , and negative pressure exerted during seroma aspiration may have contributed to ventriculoperitoneal shunt migration . excessive loose skin and increased activity may further increase the risk of migration in cats . diagnostic imaging should be offered prior to and following aspiration of shunt - associated swellings , and minimal negative pressure should be exerted . attempts to reduce the frequency of postoperative self - grooming , prevention and prompt treatment of conditions predisposing to raised intra - abdominal pressure and moderate exercise restriction , particularly within the first 3 months , may help reduce the risk of peritoneal catheter migration .

between 25% and 30% of non - small cell lung cancer ( nsclc ) patients will develop metastatic disease in the brain ( brain metastases from non - small cell lung cancer frequently they are the first site of recurrence in early - stage nsclc patients treated with definitive therapies [ 15 ] . the prognosis is poor for untreated patients with bmf - nsclc , with median overall survival ( os ) 12 months [ 1 , 4 , 5 ] . the combination of neurosurgery with stereotactic radiosurgery ( srs ) and/or whole - brain radiotherapy ( wbrt ) can increase the os up to 36 months , and in selected cases over 12 months [ 1 , 4 , 610 ] . currently , the role of systemic treatment of bmf - nsclc patients is being widely discussed [ 3 , 4 , 10 ] . historically , chemotherapy was considered as a poorly effective method of treatment , mainly because of predicted difficulties in penetrating the blood - brain - barrier ( bbb ) . for a long period of time , patients with bmf - nsclc were excluded from controlled clinical trials for chemotherapy of nsclc [ 1 , 3 , 4 , 11 , 12 ] . nowadays it seems that even if most of the drugs can not penetrate normal bbb , the integrity of the bbb is significantly altered , e.g. in bmf - nsclc patients , which can be proved by oedema and increased contrast uptake around the metastatic site . the significant amount of information indicates the possibility of efficient palliative systemic treatment of chosen patients with bmf - nsclc [ 2 , 3 , 10 , 11 , 13 ] . the role of targeted therapies , besides chemotherapy , is significantly increasing [ 1 , 4 , 10 , 13 , 14 ] . the purpose of this work is to review , relying on the literature , the actual knowledge on the methods and results of systemic treatment of brain metastases from non - small cell lung cancer . recent phase ii trials indicate efficacy , however limited , of platinum - based chemotherapy of bmf - nsclc patients [ 1520 ] , which is presented in table 1 . efficacy of platinum - based chemotherapy of bmf - nsclc patients in phase ii trials phase ii trials demonstrating efficacy of first - line bmf - nsclc chemotherapy . as outlined in table 1 , the response rates after platinum - based chemotherapy range from 23% to 45% ; chaubet - houdu and basse report 2350% . literature indicates that temozolomide ( tmz ) combined with radiotherapy , in bmf - nsclc , has a slight influence on survival , but it might increase the toxicity of the treatment [ 2 , 11 , 2123 ] . development of epidermal growth factor receptor tyrosine kinase inhibitors ( egfr - tkis ) : gefitinib or erlotinib , has clearly improved the treatment of advanced nsclc patients [ 3 , 4 , 9 , 10 , 13 , 2445 ] . egfr mutations are present in 1025% of nsclc , with the highest prevalence found in never - smoking women of east asian descent ( up to 55% ) [ 13 , 24 ] . pao et al . found egfr mutations to be present in 63% and 50% of bmf - nsclc patients , respectively , which suggests increased risk of developing brain metastases among patients with these mutations [ 25 , 26 ] . in a non - selected group of patients with bmf - nsclc the overall response rates after gefitinib range from 10% to 38% , and the duration of response ranges from 9 to 13.5 months [ 2730 ] ; erlotinib has similar efficacy [ 3135 ] . it seems that erlotinib achieves higher central nervous system ( cns ) concentration in comparison to gefitinib [ 10 , 13 ] . gefitinib and erlotinib are both approved as first - line treatment , palliative treatment ( second- and third - line ) , and in combination with radiotherapy ( wbrt srs ) , their efficacy was presented in case reports , case series , and nonrandomised phase ii trials [ 2 , 27 , 31 , 38 , 40 , 42 , 45 ] . two phase ii trials evaluated the efficacy of tki in the first - line setting on patients with bmf - nsclc [ 38 , 40 ] . both trials did not include data for egfr mutations , whereas the studies included never - smokers . reported 10 patients ; seven demonstrated an objective response to gefitinib , one had a stable disease , and two had a progressive disease after a median 48-week follow - up period . kim et al . presented a group of 23 patients with synchronous bmf - nsclc with a response rate to gefitinib or erlotinib of 69% and median overall survival of 18.8 months . the rate of cns progression was lower among egrf - mutant patients treated with gefitinib or erlotinib compared with upfront chemotherapy ( patients without egfr mutation ) 33% vs. 48% , respectively , at a median follow - up of 25 months . two phase ii trials assessed the role of gefitinib in the palliative setting in non - selected patients with bmf - nsclc [ 27 , 31 ] . reported 41 patients with a 10% response rate and median overall survival of five months , wu et al . reported 40 patients ( adenocarcinoma , never - smokers ) with a 32% response rate and median overall survival of 15 months . + gefitinib vs. wbrt + tmz , failed to show an advantage of gefitinib in a non - selected group of patients with bmf - nsclc ; os 6.3 months in the gefitinib arm and 4.9 months in the tmz arm , the difference was statistically irrelevant . . showed that tmz or erlotinib combined with wbrt + srs in a non - selected group of patients with 13 bmp - nsclc did not improve the os ; however , it increased the toxicity of the treatment . study evaluated the efficacy of erlotinib in combination with wbrt in 40 patients with bmf - nsclc . patients negative for egfr mutations had a median overall survival of 9.3 months , whereas patients positive for egfr mutations had 19.1 months . it is also undoubted that either gefitinib or erlotinib can be safely combined with wbrt [ 43 , 44 ] . some authors suggest that in selected groups of patients with bmf - nsclc , commencing treatment with gefitinib or erlotinib , with delayed wbrt , is acceptable . it relates to women with adenocarcinoma , never - smokers , and patients positive for egfr mutations . iuchi et al . presented good efficacy of gefitinib alone ( without radiotherapy ) in patients with adenocarcinoma bmf - nsclc , positive for egfr mutation the phase ii aprage trial , comparing wbrt + gefitinib with gefitinib alone in bmf - nsclc patients , is ongoing [ 3 , 12 ] . in conclusion , tki ( gefitinib , erlotinib ) overall response rate depends essentially on the presence of egfr gene activating mutation [ 10 , 12 , 13 , 36 , 37 ] ; if mutation is present , orr reaches more than 50% . in non - selected groups of patients ( adenocarcinoma , asian descents , never - smokers , asymptomatic bmf - nsclc ) after tkis therapy , it is possible to reach 70% orr [ 13 , 38 ] . tkis improve survival of bmf - nsclc patients with egfr mutations in comparison to patients without these mutations [ 10 , 12 , 13 , 39 ] . in approximately 35% of patients with nsclc , an alk ( anaplastic lymphoma kinase ) rearrangement occurs . it results in forming an eml4-alk fusion gene ; it relates to mostly young , male , never - smokers , with adenocarcinoma [ 10 , 1214 , 46 ] . in this group , administration of crizotinib , an anti - eml4-alk ( echinoderm microtubule - associated protein - like 4-anaplastic lymphoma kinase ) drug , is reasonable and effective [ 10 , 14 , 4648 ] . reported a 57% response rate , and a randomised phase iii trial presented by shaw et al . indicated statistically relevant improvement of progression - free survival of subjects treated with crizotinib , compared to patients treated with a second - line chemotherapy ( pemetrexed or docetaxel ) . unfortunately , crizotinib has a poor bbb penetration , so its efficacy in bmf - nsclc patients is doubtful [ 10 , 13 , 14 , 46 , 4951 ] . the available literature provides poor corresponding data [ 12 , 49 , 50 , 52 ] . presented a case of bmf - nsclc progression during crizotinib treatment , despite regression of the disease outside cns . . reported on crizotinib in 38 alk ( + ) patients ; 28 demonstrated progressive disease , and in 46% the first site of recurrence was bmf - nsclc . among patients with isolated recurrence in bmf - nsclc , treated with radiotherapy ( wbrt or srs ) followed by crizotinib , progression - free survival of 7.1 months single cases of bmf - nsclc responsive to crizotinib were reported by kaneda et al . and kinoshita et al . . kinoshita suggest that administering ionising radiotherapy before crizotinib treatment may play an important role in both cases [ 43 , 48 ] . in 2006 yuan et al . indicated in a murine model that cns radiotherapy increases penetrability of the bbb . mehra et al . , in a phase i trial , demonstrated responses in bmf - nsclc patients treated with one of the new generation of alk inhibitors ldk 378 . bevacizumab is a humanised monoclonal antibody that binds selectively to vegf vascular endothelial growth factor . eventually , cancer cells should not develop their own blood supply , causing a lack of oxygen and nutrients , helping to slow down the growth of the tumour . treatment of advanced nsclc with bevacizumab remains controversial [ 1 , 12 , 13 ] . results of two randomised phase iii trials , ecog 4599 and avail , reported that bevacizumab combined with chemotherapy improved the response rate and progression - free survival compared to chemotherapy alone in nsclc . ecog 4599 also reported a significantly longer os ( 12.3 vs. 10.3 months ) [ 5658 ] . hypertension , massive haemoptysis , disorders in blood coagulation , and bmf - nsclc were also qualified as exclusion criteria . the restriction of the patient population to non - squamous histology was based on the research of johnson et al . , which indicated the occurrence of life - threatening haemoptysis in this group ( 4/13 patients ) . exclusion of bmf - nsclc patients was based on the current opinion that bevacizumab significantly increase the risk of intracranial bleeding in this group [ 1 , 12 , 13 ] . in both trials , the incidence of cns haemorrhages among patients receiving bevacizumab was similar to the incidence of those reported in patients who did not receive bevacizumab . based on the results of these trials , bevacizumab is currently licensed for use ase first - line therapy in combination with chemotherapy ( carboplatin + paclitaxel ) in the usa , or in addition to platinum - based chemotherapy in europe in patients with advanced non - squamous nsclc . however , it does not mean it is commonly used ; this is because of absent or poor benefit compared to chemotherapy alone , with a slightly increased toxicity . it is obvious that there are no reasons to exclude patients with brain metastases from clinical trials on antiangiogenic agents , as took place in the recent past [ 1 , 13 , 56 ] . despite antiangiogenic therapy , patients with or without brain metastases have similar risk of intracranial bleeding ( 90.83.3% ) [ 6064 ] . several retro- and prospective clinical trials conducted in the past few years indicate that the combination of bevacizumab with chemotherapy or erlotinib is safe in the treatment of bmf - nsclc , with a slight risk of intracranial bleeding [ 60 , 6268 ] . a prospective phase iv study aries evaluated the safety and efficacy of the first - line setting in patients with non - squamous nsclc treated with bevacizumab combined with chemotherapy . a total of 150 patients with bmf - nsclc were enrolled , median pfs and os were 6.0 and 11.7 months , respectively , and no grade 3 to 5 cns haemorrhage occurred . the phase ii study passport enrolled 115 nsclc patients with previously treated bmf - nsclc with wbrt and/or surgery . patients received as a first - line bevacizumab , with platinum - based doublet chemotherapy or erlotinib , and as a second - line , bevacizumab with single - agent chemotherapy or erlotinib ; no grades 1 to 5 cns haemorrhage , among patients who received bevacizumab - based therapy were reported . the phase iii atlas study was designed to evaluate the combination of bevacizumab / erlotinib versus bevacizumab / placebo as maintenance therapy after four cycles of induction platinum - containing chemotherapy plus bevacizumab as first - line treatment in advanced nsclc patients . among 25 evaluable patients with a history of cns metastases pretreated with wbrt and/or neurosurgery , one grade 2 cns bleeding was observed in a patient on post - progression therapy after 14 cycles of bevacizumab [ 66 , 67 ] . the sail study assessed the safety and efficacy of the addition of bevacizumab to first - line chemotherapy . this study proved that bevacizumab - based therapy resulted in median os of 14.6 months , with a median time to disease progression of 7.8 months . the specific safety of bevacizumab was assessed in patients who either developed bmf - nsclc during treatment or had occult bmf - nsclc at study entry . of the 281 patients evaluated , five ( 2% ) had cns bleeding . the phase iii betalung study evaluated the addition of bevacizumab to erlotinib for the second - line treatment of advanced nsclc patients . a total of 636 patients were randomised to receive bevacizumab in combination with either erlotinib or erlotinib alone . the addition of bevacizumab to erlotinib increased pfs compared to erlotinib alone ( 3.4 vs. 1.7 months , respectively ) . this trial included patients with bmf - nsclc , previously treated with wbrt and neurosurgery or wbrt + srs . among 68 bmf - nsclc patients , 37 received erlotinib + bevacizumab and 31 erlotinib alone . besse et al . presented an analysis including more than 12,000 advanced / metastatic breast cancer , nsclc , renal , and colorectal cancer patients , with previously treated cns metastases , from 13 phase ii / iii randomised controlled trials , two open - label , single - arm safety studies , and two prospective studies . the rate of cerebral haemorrhage in the bevacizumab - treated group was 3.3% , compared to 1% in the group not treated with bevacizumab . this study suggests that the administration of bevacizumab should no longer be contraindicated based solely on the presence of cns metastases . several clinical trials have been launched to determine the safety and efficacy of various other antiangiogenic agents in the treatment of new or progressive brain metastases from solid tumours : sunitinib , cediranib , and vatalanib . chemotherapy is generally effective in bmf - nsclc , and platinum - based provides response rates ranging from 23% to 45% . epidermal growth factor receptor tyrosine kinase inhibitors ( egfr tkis ) gefitinib and erlotinib have a definite activity in bmf - nsclc with activating egfr mutation , or in selected groups of patients ( woman of east asian descent , never - smokers , those with adenocarcinoma ) ; the response rate ranges from 38% to 70% . both egfr - tkis have been investigated in first - line , palliative , and in combination with radiotherapy . patients with bmf - nsclc - egfr - mutant have improved overall survival compared with egfr wild - type tumours , when receiving an egfr inhibitor . there is no data on the activity of the agent ani - eml4-alk - crizotinib in patients with bmf - nsclc . data from a clinical trial enrolling patients with pretreated or occult bmf - nsclc showed that the addition of bevacizumab to various chemotherapy agents or erlotinib is a safe and efficient treatment , associated with a low incidence of cns haemorrhage . however , bevacizumab should be used with caution in patients with active bmf - nsclc . many organs can be herniated into the scrotum such as small intestine , appendix , colon , and ovaries . ureteral herniation is extremely rare , usually asymptomatic and is reported as isolated case report or small series . we report the case of an 88-year - old man treated for inguinoscrotal hernia where the left ureter was incidentally found in the herniated retroperitoneal fat . presenting symptoms , diagnostic evaluation , and surgical management an 88-year - old man was admitted for a left moderately sized inguinoscrotal hernia . his medical history included hypertension and benign prostatic hyperplasia . the herniated parts were dislocated from the scrotum , as well as the testicle , and cord strictures . the cord was separated from the herniated parts and was partially covered from preperitoneal fat . a large amount of retroperitoneal fat surrounded from a sac - like formation was found adjacent to the cordis [ figure 1a and b ] . these adhesions , as well as a part of this fat , were excised because of the irreducibility of the mass . during the excision , 24 h later , an abdominal ultrasound was performed to rule out any structural abnormality , which may have been missed preoperatively . ( a and b ) the left extraperitoneal inguinoscrotal hernia the ureter identified into the herniated retroperitoneal fat ureteral herniation is rare , and approximately 140 cases have been reported in the literature , mainly as isolated case reports or small series . ureteric hernia is also reported as spontaneous , postoperative or as a complication of renal transplantation . ureteral herniation presents as a groin mass usually asymptomatic although many cases describe association with dysuria , hematuria , and hydronephrosis . the hernia has the classical aspect of an indirect hernia ; it is formed by the sac anteromedially that contain viscera that make up the wall of the sac , and the ureter lying posterolaterally . the ureter slides into the canal drawn by the posterior peritoneum that follows the herniated viscera . paraperitoneal hernias are more common in men , usually located on the right side , are often large in size , usually reducible and rarely symptomatic . there is not a clear association with kidney or ureteric abnormalities . on the other hand , the hernia is often accompanied by the large amount of retroperitoneal fat , is often nonreducible , usually small and commonly associated with urinary symptoms . authors consider this type of hernia congenital and related to developmental abnormalities of differentiation of the ureter from the wolffian duct . the ureter slides along with the testis into the scrotum ; a process also favored by adhesions between the ureter and genitoinguinal ligaments . many extraperitoneal hernias have a congenital association to renal or ureteral malformation such as crossed renal ectopia or nephroptosis . however , in our case the hernia was sizable , partially reducible and was no associated with urinary symptoms . the diagnosis of ureteral hernia is often missed due to lack of urinary symptoms or signs or symptoms that could lead doctors to apply an extended preoperative work up . although computed tomography may determine the type and the contents of hernias and the intravenous urography may determine abnormalities before surgery , they are not justifiable on every inguinal hernia repair . considering the fact that ureteric injuries are serious complications and require additional surgical approaches , we emphasize the high index of suspicion needed by surgeons when repairing hernias identifying gross amount of sliding fat and resecting fat trying to reduce hernia into the abdomen .

in the systemic treatment of brain metastases from non - small cell lung cancer ( bmf - nsclc ) chemo- and targeted therapy are used . response rates after platinum - based chemotherapy , range from 23% to 45% . development of epidermal growth factor receptor tyrosine kinase inhibitors ( egfr - tkis ) : gefitinib or erlotinib , was an improvement in treatment of advanced nsclc patients . egfr mutations are present in 1025% of nsclc ( mostly adenocarcinoma ) , and up to 55% in never - smoking women of east asian descent . in the non - selected group of patients with bmf - nsclc , the overall response rates after gefitinib or erlotinib treatment range from 10% to 38% , and the duration of response ranges from 9 to 13.5 months . in the case of present activating egfr mutation , the response rate after egrf - tkis is greater than 50% , and in selected groups ( adenocarcinoma , patients of asian descent , never - smokers , asymptomatic bmf - nsclc ) even 70% . gefitinib or erlotinib treatment improves survival of bmf - nsclc patients with egfr mutation in comparison to cases without the presence of this mutation . there is no data on the activity of the anti - eml4-alk agent crizotinib . bevacizumab , recombinant humanised monoclonal antibody anti - vegf , in the treatment of advanced non - squamous nsclc patients is a subject of intense research . data from a clinical trial enrolling patients with pretreated or occult bmf - nsclc proved that the addition of bevacizumab to various chemotherapy agents or erlotinib is a safe and efficient treatment , associated with a low incidence of csn haemorrhages . however , the efficacy and safety of bevacizumab used for therapeutic intent , regarding active brain metastases is unknown . an inguinoscrotal hernia is a common disorder that usually contains intraperitoneal organs ( small intestine , colon , appendix , ovaries ) . extraperitoneal ureteral herniation into an inguinoscrotal hernia is a rare condition and often associated with congenital abnormalities or postoperative anatomic changes . a high index of suspicion is needed in order to avoid intraoperative ureteric injuries . we herein report the case of a ureteric herniation into an inguinoscrotal hernia incidentally found during a scheduled hernia repair .

the incidence of non - hodgkin lymphoma ( nhl ) is 100200-fold higher in hiv - infected individuals than in the general population . nhl is an aids - defining illness and in 35% of cases , the lymphoma is the initial manifestation of aids . the most common hiv - associated lymphomas are diffuse large b cell lymphoma ( dlbcl ; often primary in the central nervous system ) , burkitt lymphoma , primary effusion lymphoma , and plasmablastic lymphoma of the oral cavity . primary mediastinal large b cell lymphoma is a subtype of diffuse large b cell lymphoma arising in the mediastinum with distinctive clinical , morphologic , and genotypic features . most patients are young adult females presenting with localized mediastinal mass and symptoms often related to local effects of the large mediastinal tumor such as superior vena cava syndrome . dlbcl is one of the most common lymphomas in the setting of hiv , but the incidence of pmlbcl in hiv is not well established . only one reported case of hiv - associated pmlbcl has been found in the english literature . a 25-year - old female presented to her physician with 1 week of worsening shortness of breath , cough , and chest pain . she gave birth to her fourth child 5 weeks ago , and her cough and shortness of birth was developed a few days prior to delivery . she was diagnosed with hiv 1 year before ; however , she had no previous manifestation of aids . chest computed tomography ( ct ) revealed a 13.7 9.2-cm superior mediastinal mass , multiple left pleural masses ranging from 2.42.7 cm , a 1.6-cm left upper lobe lung nodule , and bilateral pleural effusions ( fig . 1 ) . the infiltrating cells were mostly large with irregular and convoluted vesicular nuclei , 13 visible nucleoli , and moderately abundant cytoplasm . the tumor cells were cd45 + , cd20 + , cd23 + , partially cd30 + , weak partial positive for bcl-2 and bcl-6 , and negative for cd3 , cd5 , cd10 , cd15 , cd21 , and alk-1 . eber in situ hybridization was positive in approximately 30% of the tumor cells ( fig . 2 ) . flow cytomeric immunophenotyping showed a decreased cd4/cd8 ratio of 0.3 and evidence of an abnormal b cell population with expression of cd19 and cd20 but lack of surface immunoglobulin light chain expression . lumbar puncture and a magnetic resonance imaging of the brain showed no evidence of central nervous system involvement of lymphoma . her symptoms initially improved ; however , after the third cycle , her disease progressed and the treatment was changed to vp-16 , ifosfamide , and ara - c with concurrent mesna . her symptoms continued worsening and she died of respiratory failure 5 months after the diagnosis of the lymphoma . 1chest ct of case 1 reveals a 13.7 9.2-cm superior mediastinal mass , multiple left pleural masses ranging from 2.42.7 cm , a 1.6-cm left upper lobe lung nodule , and bilateral pleural effusionsfig . 2pathologic examination of case 1 shows varying degrees of compartmentalizing fibrosis associated with lymphocytic infiltrate . the infiltrating cells are mostly large with irregular and convoluted vesicular nuclei , 13 visible nucleoli , and moderate cytoplasm . chest ct of case 1 reveals a 13.7 9.2-cm superior mediastinal mass , multiple left pleural masses ranging from 2.42.7 cm , a 1.6-cm left upper lobe lung nodule , and bilateral pleural effusions pathologic examination of case 1 shows varying degrees of compartmentalizing fibrosis associated with lymphocytic infiltrate . the infiltrating cells are mostly large with irregular and convoluted vesicular nuclei , 13 visible nucleoli , and moderate cytoplasm . eber in situ hybridization is positive in approximately 30% of the tumor cells a 38-year - old male with history of hiv for 7 years and no prior aids - defining illness presented with complaints of progressive shortness of breath for 10 days and one episode of fever 2 weeks prior to the onset of his symptoms . he had night sweats for about 3 years , but he denied weight loss , cough , or hemoptysis . on admission , his laboratory values showed a cd4 count of 140/l and a markedly elevated ldh value of 886 . imaging studies of the chest revealed an abnormal cardiomediastinal silhouette caused by a large left - sided mediastinal mass extending within the superior hemithorax medially . in addition , large left - sided pleural effusion was present , and several nodular opacities were noted in the left lung ( fig . 3 ) . the patient was admitted , and highly active antiretroviral therapy ( haart ) was started . the biopsy showed a variably dense lymphoid infiltrate composed of large lymphoid cells with abundant pale cytoplasm , pleomorphic nuclei which focal clusters , and rounded nests surrounded by delicate fibrous bands . immunohistochemistry showed that the tumor cells were positive for cd45 , cd20 , cd30 , bcl-2 , and bcl-6 , were focally positive for cd23 , and were negative for cytokeratin , cd3 , cd10 , cd15 , and alk-1 . eber in situ hybridization was mostly negative , with only very rare scattered positively staining cells ( fig . flow cytometric analysis showed a population of t lymphocytes with a cd4/cd8 ratio of 1.4 , but no discrete cd19 + or cd20 + b cell cluster was identified . a positron emission tomography scan , however , revealed multifocal extranodal thoracic , both nodal and extranodal abdominal , and possible acetabular involvement by the lymphoma . the patient was treated with steroids followed by combination chemotherapy with rituxan - hypercvad / intrathecal metothrexate in addition to haart and antimicrobial medication including bactrim , acyclovir , and diflucan . after 2 cycles of therapy , the mediastinal mass decreased in size , and the ldh level dropped to 168 . the patient responded well to the therapy and after 9 months of follow - up , he is without detectable disease . 3chest x - ray of case 2 shows an abnormal cardiomediastinal silhouette caused by a large left - sided mediastinal mass extending within the superior hemithorax medially . in addition , large left - sided pleural effusion is present , and several nodular opacities are noted in the left lungfig . 4microscopic sections of case 2 show dense lymphoid infiltrate composed of large lymphoid cells with abundant pale cytoplasm , pleomorphic nuclei which form clusters and rounded nests surrounded by delicate fibrous bands . eber in situ hybridization was negative chest x - ray of case 2 shows an abnormal cardiomediastinal silhouette caused by a large left - sided mediastinal mass extending within the superior hemithorax medially . in addition , large left - sided pleural effusion is present , and several nodular opacities are noted in the left lung microscopic sections of case 2 show dense lymphoid infiltrate composed of large lymphoid cells with abundant pale cytoplasm , pleomorphic nuclei which form clusters and rounded nests surrounded by delicate fibrous bands . a 25-year - old female presented to her physician with 1 week of worsening shortness of breath , cough , and chest pain . she gave birth to her fourth child 5 weeks ago , and her cough and shortness of birth was developed a few days prior to delivery . she was diagnosed with hiv 1 year before ; however , she had no previous manifestation of aids . chest computed tomography ( ct ) revealed a 13.7 9.2-cm superior mediastinal mass , multiple left pleural masses ranging from 2.42.7 cm , a 1.6-cm left upper lobe lung nodule , and bilateral pleural effusions ( fig . 1 ) . the infiltrating cells were mostly large with irregular and convoluted vesicular nuclei , 13 visible nucleoli , and moderately abundant cytoplasm . the tumor cells were cd45 + , cd20 + , cd23 + , partially cd30 + , weak partial positive for bcl-2 and bcl-6 , and negative for cd3 , cd5 , cd10 , cd15 , cd21 , and alk-1 . eber in situ hybridization was positive in approximately 30% of the tumor cells ( fig . 2 ) . flow cytomeric immunophenotyping showed a decreased cd4/cd8 ratio of 0.3 and evidence of an abnormal b cell population with expression of cd19 and cd20 but lack of surface immunoglobulin light chain expression . lumbar puncture and a magnetic resonance imaging of the brain showed no evidence of central nervous system involvement of lymphoma . her symptoms initially improved ; however , after the third cycle , her disease progressed and the treatment was changed to vp-16 , ifosfamide , and ara - c with concurrent mesna . her symptoms continued worsening and she died of respiratory failure 5 months after the diagnosis of the lymphoma . 1chest ct of case 1 reveals a 13.7 9.2-cm superior mediastinal mass , multiple left pleural masses ranging from 2.42.7 cm , a 1.6-cm left upper lobe lung nodule , and bilateral pleural effusionsfig . 2pathologic examination of case 1 shows varying degrees of compartmentalizing fibrosis associated with lymphocytic infiltrate . the infiltrating cells are mostly large with irregular and convoluted vesicular nuclei , 13 visible nucleoli , and moderate cytoplasm . chest ct of case 1 reveals a 13.7 9.2-cm superior mediastinal mass , multiple left pleural masses ranging from 2.42.7 cm , a 1.6-cm left upper lobe lung nodule , and bilateral pleural effusions pathologic examination of case 1 shows varying degrees of compartmentalizing fibrosis associated with lymphocytic infiltrate . the infiltrating cells are mostly large with irregular and convoluted vesicular nuclei , 13 visible nucleoli , and moderate cytoplasm . a 38-year - old male with history of hiv for 7 years and no prior aids - defining illness presented with complaints of progressive shortness of breath for 10 days and one episode of fever 2 weeks prior to the onset of his symptoms . he had night sweats for about 3 years , but he denied weight loss , cough , or hemoptysis . on admission , his laboratory values showed a cd4 count of 140/l and a markedly elevated ldh value of 886 . imaging studies of the chest revealed an abnormal cardiomediastinal silhouette caused by a large left - sided mediastinal mass extending within the superior hemithorax medially . in addition , large left - sided pleural effusion was present , and several nodular opacities were noted in the left lung ( fig . 3 ) . the patient was admitted , and highly active antiretroviral therapy ( haart ) was started . the biopsy showed a variably dense lymphoid infiltrate composed of large lymphoid cells with abundant pale cytoplasm , pleomorphic nuclei which focal clusters , and rounded nests surrounded by delicate fibrous bands . immunohistochemistry showed that the tumor cells were positive for cd45 , cd20 , cd30 , bcl-2 , and bcl-6 , were focally positive for cd23 , and were negative for cytokeratin , cd3 , cd10 , cd15 , and alk-1 . eber in situ hybridization was mostly negative , with only very rare scattered positively staining cells ( fig . flow cytometric analysis showed a population of t lymphocytes with a cd4/cd8 ratio of 1.4 , but no discrete cd19 + or cd20 + b cell cluster was identified . a positron emission tomography scan , however , revealed multifocal extranodal thoracic , both nodal and extranodal abdominal , and possible acetabular involvement by the lymphoma . the patient was treated with steroids followed by combination chemotherapy with rituxan - hypercvad / intrathecal metothrexate in addition to haart and antimicrobial medication including bactrim , acyclovir , and diflucan . after 2 cycles of therapy , the mediastinal mass decreased in size , and the ldh level dropped to 168 . the patient responded well to the therapy and after 9 months of follow - up , he is without detectable disease . 3chest x - ray of case 2 shows an abnormal cardiomediastinal silhouette caused by a large left - sided mediastinal mass extending within the superior hemithorax medially . in addition , large left - sided pleural effusion is present , and several nodular opacities are noted in the left lungfig . 4microscopic sections of case 2 show dense lymphoid infiltrate composed of large lymphoid cells with abundant pale cytoplasm , pleomorphic nuclei which form clusters and rounded nests surrounded by delicate fibrous bands . eber in situ hybridization was negative chest x - ray of case 2 shows an abnormal cardiomediastinal silhouette caused by a large left - sided mediastinal mass extending within the superior hemithorax medially . in addition , large left - sided pleural effusion is present , and several nodular opacities are noted in the left lung microscopic sections of case 2 show dense lymphoid infiltrate composed of large lymphoid cells with abundant pale cytoplasm , pleomorphic nuclei which form clusters and rounded nests surrounded by delicate fibrous bands . the world health organization classification of hematopoietic malignancies recognizes aids - associated lymphomas as a specific category to acknowledge the significant impact of hiv on the pathophysiology and prognosis of lymphomas . the pathogenetic mechanisms contributing to lymphoma genesis are complex and include chronic antigen stimulation , genetic abnormalities , cytokine dysregulation , and the role of herpes viruses : epstein barr virus ( ebv ) and kaposi sarcoma human virus ( kshv , hhv8 ) . the overall survival of patients with hiv - related nhls is inferior compared to hiv - negative patients , and in hiv , the majority of patients present with widespread disease and with higher risk for central nervous system involvement . the introduction of haart extended the lifespan of aids patients and some studies indicated a decrease of the incidence of lymphomas in aids since widespread availability of haart . the combination of haart with aggressive chemotherapy , such as chop , not only is tolerable and effective treatment in patients with hiv and nhl but the initial good response to haart is also associated with higher complete response rates to the chemotherapy ( 77% response to chop versus 50% in nonresponders ) . the association between hiv and classical hodgkin lymphoma is less clear , and , unexpectedly , the incidence of hodgkin lymphoma in aids has increased since haart was introduced . of note , hiv - related hodgkin lymphoma is associated with ebv in nearly all cases , and it has been postulated that the ebv - encoded latent membrane protein ( lmp1 ; which is functionally homologous to activated cd40 ) , is essential to the antiapoptotic phenotype of the reed sternberg cells , replacing the cd40/cd40 ligand interaction by cd4 + t cells , which are decreased in aids . though diffuse large b cell lymphoma is one of the most common nhl associated with aids , so far , only one case of pmlbcl has been described in the literature in the setting of aids . this patient , a 29-year - old woman with 12 years of history of hiv infection without prior aids - defining illness , presented with fever , night sweats , cough , and a cd4 count of 316/l . haart therapy was initiated but had to be discontinued within a few days due to development of stevens johnson syndrome . biopsies via mediastinotomy established the diagnosis of pmlbcl , with no evidence of extrathoracic disease upon staging . chemotherapy with chop and granulocyte colony - stimulating factor was started ; however , 6 days after the first cycle of treatment , the patient died from neutropenic sepsis . in our current report , the clinical presentation in case 1 is classical in terms that the patient is a young woman who presents with a mediastinal mass and her disease is localized to the mediastinum ( table 1 summarizes the clinical findings of cases 1 and 2 in comparison to the case reported by milling et al . ) . this patient was known to have hiv but because she was pregnant , she only received azt monotherapy and had very low cd4 t - cell count ( 152/l ) at diagnosis . in contrast , the clinical presentation of case 2 is unusual in several aspects . the patient is a male and in addition to a large mediastinal mass , he has widespread disease at presentation . similarly to case 1 , he did not receive haart therapy prior to his presentation and his cd4 t - cell count was low ( 140/l ) . neither of the two patients had evidence of bone marrow or central nervous system involvement . the immunophenotype of the lymphoma cells of both cases is consistent with previously described phenotype of pmlbcl : cd45 + , cd20 + , cd23 + , and partially cd30 + . in case 2 , but not in case 1 , the lymphoma cells also express bcl-6 , a finding unusual but not excluding the diagnosis of pmlbcl . an important finding in case 1 is the ebv positivity in approximately 30% of the lymphoma cells by eber in situ hybridization . the role of ebv in the pathogenesis of pmlbcl in the immunocompetent host was previously dismissed . in one study , only two cases were positive for ebv of 41 pmlbcl cases ; others reported complete absence of ebv . the previously reported case of pmlbcl in hiv - infected patient also was negative for ebv with eber in situ hybridization . in our case , it is not clear as to what extent the ebv positivity contributes to lymphoma pathogenesis or represents ebv infection acquired during the course of disease . yet , the finding of 30% eber - positive lymphoma cells is likely to be clinically significant , since park et al . recently reported that eber positivity of 20% or more in lymphoma cells in dlbcl is associated with significantly poorer overall survival and progression - free survival compared to ebv - negative dlbcls . in the immunocompromised , such as aids or post - transplant state , ebv infection even in a minority of lymphoma cells might have significant impact on disease pathogenesis and outcome . since the molecular signature of pmlbcl resembles that of classical hodgkin lymphoma , ebv infection might contribute to an anti - apoptotic phenotype of the lymphoma cells with a similar mechanism as observed in hiv - associated hodgkin lymphoma . in our patient with ebv - associated pmlbcl , despite the localized nature of the lymphoma , the tumor continued to progress during chemotherapy resulting in death of the patient after 5 months of diagnosis . unlike case 1 , case 2 responded well to a combination of haart and chemotherapy and improved rapidly , despite widespread disease at diagnosis . table 1comparison of the clinical features of case 1 , case 2 , and the case reported by milling et al . 2004age / sex25/female38/male29/femalepresentationshortness of breath , cough , chest painshortness of breath , fever , night sweatsfever , night sweats and coughduration of hiv+ status1 year7 years12 yearsprior therapy for hivazathyoprinenonenonecd4 count at presentation152/l140/l316/lldh at presentation379 iu / l ( high)886 iu / l ( high)542 iu / l ( high)stage of diseasestage ivstage ivnot statedbone marrow involvementnegativenegativenegativeextramediastinal involvementpleural masses , pleural effusions , left lung nodulepleural effusion , left lung nodules , multifocal abdominal and acetabular massesnonetherapy receivedhaart , 3 cycles of epoch then vp-16 , ifosfamide , ara - c , mesnahaart , 6 cycles of rituxan - hyper cvad1 cycle of chop and g - csfoutcomedeath 5 months after diagnosisno detectable disease after 9 months follow - updeath 6 days after chemotherapy comparison of the clinical features of case 1 , case 2 , and the case reported by milling et al . 2004 in summary , though increased incidence of pmlbcl has not been established in aids due to the rarity of these cases , pmlbcl occurs in hiv and appears to present in the setting of low cd4 t - cell count as a rapidly growing mediastinal mass . a good response to a combination of haart and chemotherapy more cases are necessary to characterize the relationship between pmlbcl and hiv and to determine the impact of ebv infection on disease pathogenesis and prognosis in the immunocompromised .

in the closed structure of the p2x cation channel , three -helical transmembrane domains cross the membrane obliquely : in rat p2x2 receptors , these intersect at thr339 . replacing thr339 by lysine in one , two or three subunits progressively increased chloride permeability and reduced unitary conductance . this implies that the closed - open transition involves a symmetrical separation of the three subunits , and that thr339 from each contributes symmetrically to the open channel permeation pathway . primary mediastinal large b cell lymphoma ( pmlbcl ) is a subtype of diffuse large b cell lymphoma arising in the mediastinum with distinctive clinical and morphological features . though diffuse large b cell lymphoma is one of the most common non - hodgkin lymphoma associated with aids , there are no data available regarding the association of hiv and pmlbcl . we report here two cases of pmlbcl arising in aids patients . in both cases , pmlbcl presented in a setting of low cd4 t - cell count as rapidly enlarging mediastinal mass . the morphologic and immunophenotypic findings are characteristic of pmlbcl . one of the two patients , a 25-year - old woman who had localized disease and evidence of epstein barr virus in lymphoma cells , did not respond to chemotherapy and died of disease progression 5 months after diagnosis . the second patient , a 38-year - old male with disseminated disease , responded to therapy and is disease - free after 9 months of follow - up .

cardiovascular disease ( cvd ) is the main cause of death in maintenance hemodialysis ( mhd ) patients . both aortic artery calcification ( aac ) and cardiac valve calcification ( cvc ) have a high incidence in dialysis patients . the diagnosis of vascular calcification is usually based on very expensive and highly technical devices such as electron beam computed tomography ( ebct ) or multislice spiral computed tomography ( ct ) . however , lateral lumbar x - ray is a useful approach to detect aac with cheap price and low radiation . in addition , the use of plain radiographic films of bone has already been suggested in kidney disease improve global outcomes ( kdigo ) chronic kidney disease mineral and bone disorder ( ckd - mbd ) clinic practice guideline . our previous studies have already showed the high incidence of aac and cvc in dialysis patients and increased fgf23 ( fibroblast growth factor 23 ) was associated with aac and cvc . now , in this study , we aimed to investigate the relationship among aac , cvc , and mortality , and to figure out that , which factor could predict the outcome of mhd patients . two hundred forty - seven mhd patients were treated in ruijin hospital affiliated to shanghai jiao tong university , school of medicine in july 2011 . two hundred seventeen patients met the following inclusion criteria : ( 1 ) age over 18 years , ( 2 ) patients received hemodialysis 3 times a week , on a 4 h schedule , using a dialysate calcium concentration of 1.5 mmol / l , ( 3 ) no rapidly progressive kidney disease . among these patients , 74 patients refused to take part in this study , 18 patients with cancer , 15 patients dialysis vintage less than 3 months . this study was approved by the institutional review board of the ruijin hospital , shanghai jiao tong university , school of medicine and was in accordance with the principle of the helsinki declaration . all clinic data of mhd patients were collected , including blood pressure , which were recorded using the mean of the previous 1-month , height and weight , and medical history . predialysis blood tests were collected , which include prealbumin , alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , total protein , albumin , blood urea nitrogen , serum creatinine , uric acid , parathyroid hormone ( pth ) , 25-hydroxy vitamin d ( 25(oh)d ) , triglyceride , cholesterol , high density lipoprotein , low density lipoprotein , serum phosphate , and calcium . pth was measured using an intact assay by a chemiluminescent method ( abbott i2000 ) ; serum 25(oh)d was measured by electrochemiluminescence immunoassay ( roche cobas e601 ) . we collected all the samples with other blood test samples on the same day in july 2011 before dialysis . after centrifugation for 10 min at 2000 rpm , all plasmas were stored at 80c as soon as possible . plasma fgf23 level was measured using a c - terminal assay ( fgf23 [ c - term ] elisa , immutopics inc . body mass index was calculated as weight in kilograms divided by height in meters squared . all echocardiographic measurements were performed according to the recommendations of the american society of echocardiography by 2 sonographers unaware of biochemical results . two - dimensional assessment of the aortic valve and mitral valve , together with continuous - wave doppler ultrasonography , was performed according to parasternal long - axis and short - axis views . cvc is defined as bright echoes of more than 1 mm on 1 or more cusps of the aortic or mitral valve or mitral annulus . aac was detected by a lateral lumbar x - ray plain radiography at a voltage of 70 kv in 120 mhd patients and read by two radiologists using a semi - quantitative score [ figure 1 ] . this semi - quantitative score also used by others and summarized as follows : calcified deposits along the anterior and posterior longitudinal walls of the abdominal aorta adjacent to each lumbar vertebra from l1 to l4 were assessed using the midpoint of the intervertebral space above and below the vertebrae as the boundaries . calcifications were graded as follows : 0 , no aortic calcific deposits ; 1 , less than one - third of the corresponding vertebral length ; 2 , one - third or more , but less than two - thirds of the corresponding length ; 3 , more than two - thirds of corresponding length . each patient 's radiological semi - quantitative score ranged from 0 to 4 for segment affected , 06 for each vertebral level , and 024 for the total score . statistical package for social analysis ( spss for windows , ibm corp , usa ) version 19.0 was used for data analysis . results are expressed as mean standard error of mean , median ( and range ) , or frequency ( as percentage ) . comparison between groups was performed by an unpaired t - test or the nonparametric wilcoxon rank sum test in case of nonnormally distributed variables . the kaplan meier method was used to estimate survival probabilities using the log - rank test . two hundred forty - seven mhd patients were treated in ruijin hospital affiliated to shanghai jiao tong university , school of medicine in july 2011 . two hundred seventeen patients met the following inclusion criteria : ( 1 ) age over 18 years , ( 2 ) patients received hemodialysis 3 times a week , on a 4 h schedule , using a dialysate calcium concentration of 1.5 mmol / l , ( 3 ) no rapidly progressive kidney disease . among these patients , 74 patients refused to take part in this study , 18 patients with cancer , 15 patients dialysis vintage less than 3 months . this study was approved by the institutional review board of the ruijin hospital , shanghai jiao tong university , school of medicine and was in accordance with the principle of the helsinki declaration . all clinic data of mhd patients were collected , including blood pressure , which were recorded using the mean of the previous 1-month , height and weight , and medical history . predialysis blood tests were collected , which include prealbumin , alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , total protein , albumin , blood urea nitrogen , serum creatinine , uric acid , parathyroid hormone ( pth ) , 25-hydroxy vitamin d ( 25(oh)d ) , triglyceride , cholesterol , high density lipoprotein , low density lipoprotein , serum phosphate , and calcium . pth was measured using an intact assay by a chemiluminescent method ( abbott i2000 ) ; serum 25(oh)d was measured by electrochemiluminescence immunoassay ( roche cobas e601 ) . we collected all the samples with other blood test samples on the same day in july 2011 before dialysis . after centrifugation for 10 min at 2000 rpm , all plasmas were stored at 80c as soon as possible . plasma fgf23 level was measured using a c - terminal assay ( fgf23 [ c - term ] elisa , immutopics inc . body mass index was calculated as weight in kilograms divided by height in meters squared . all echocardiographic measurements were performed according to the recommendations of the american society of echocardiography by 2 sonographers unaware of biochemical results . two - dimensional assessment of the aortic valve and mitral valve , together with continuous - wave doppler ultrasonography , was performed according to parasternal long - axis and short - axis views . cvc is defined as bright echoes of more than 1 mm on 1 or more cusps of the aortic or mitral valve or mitral annulus . aac was detected by a lateral lumbar x - ray plain radiography at a voltage of 70 kv in 120 mhd patients and read by two radiologists using a semi - quantitative score [ figure 1 ] . this semi - quantitative score also used by others and summarized as follows : calcified deposits along the anterior and posterior longitudinal walls of the abdominal aorta adjacent to each lumbar vertebra from l1 to l4 were assessed using the midpoint of the intervertebral space above and below the vertebrae as the boundaries . calcifications were graded as follows : 0 , no aortic calcific deposits ; 1 , less than one - third of the corresponding vertebral length ; 2 , one - third or more , but less than two - thirds of the corresponding length ; 3 , more than two - thirds of corresponding length . each patient 's radiological semi - quantitative score ranged from 0 to 4 for segment affected , 06 for each vertebral level , and 024 for the total score . the blood tests and x - ray plain radiography statistical package for social analysis ( spss for windows , ibm corp , usa ) version 19.0 was used for data analysis . results are expressed as mean standard error of mean , median ( and range ) , or frequency ( as percentage ) . comparison between groups was performed by an unpaired t - test or the nonparametric wilcoxon rank sum test in case of nonnormally distributed variables . the kaplan meier method was used to estimate survival probabilities using the log - rank test . totally , 110 mhd patients were involved in this study . of whom , 64 ( 58.2% ) patients were male , the mean age was 55.2 1.4 years , and the median dialysis duration was 29.85 ( 3.0225.5 ) months . the demographic and clinical characteristics of mhd patients are shown in table 1 . among 110 mhd patients , only one patient received parathyroidectomy and 15 ( 13.6% ) patients had diabetes mellitus . clinical characteristics of mhd patients ( n = 110 ) * our previous study has shown that fgf23 level in general population is < 100 ru / ml , which is consistent with other study ; reference range according to kdigo ckd - mbd guideline . bmi : body mass index ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; alp : alkaline phosphatase ; tp : total protein ; bun : blood urea nitrogen ; scr : serum creatinine ; ua : uric acid ; ipth : intact parathyroid hormone ; 25(oh)d : 25-hydroxy vitamin d ; tg : triglyceride ; tc : total cholesterol ; hdl : high density lipoprotein ; ldl : low density lipoprotein ; fgf23 : fibroblast growth factor 23 ; p : phosphate ; ca : calcium ; mhd : maintenance hemodialysis ; alb : albumin ; ckd - mbd : chronic kidney disease mineral and bone disorder ; kdigo : kidney disease improve global outcomes : improving global outcomes . : not available . twenty - eight ( 25.5% ) of 110 mhd patients had cvc from echocardiography , 25 ( 22.7% ) with aortic valve calcification , 10 ( 9.1% ) with mitral valve calcification , and only 1 ( 0.9% ) with tricuspid valve calcification . sixteen ( 25.0% ) of male and 12 ( 26.1% ) of female patients have cvc in our study . there is no gender difference in the incidence of cvc ( p = 0.897 ) . sixty - eight ( 61.8% ) of 110 mhd patients had visible calcification of aorta from lateral lumbar x - ray plain radiography , and the mean involved segments were 1.59 with mean aacs 4.21 0.51 scores . in analysis of the incidence of each segment of aac , l1 segment was 25.5% , l2 41.8% , l3 42.7% and l4 49.1% . thirty - eight ( 59.4% ) of male and 30 ( 65.2% ) of female have aac . there is no gender difference in the incidence of aac ( p = 0.534 ) . after 42 months follow - up , 25 ( 22.7% ) patients died , including 16 cases from cardiovascular events , 6 respiratory failure , 2 abandon treatment , and 1 deep venous thrombosis of lower extremity . in our study , 19 ( 29.7% ) male patients and 6 ( 13.0% ) female patients died during follow - up . meier analyses were performed to examine the univariate association between the presence of abdominal aortic calcification , cvc , and outcome . figure 2 shows the relationship among aac , cvc , and death from all - causes mortality and cardiovascular mortality . patients with aac had a significantly greater number of deaths from all - cause than those without aac ( log - rank test , p = 0.002 ) . similarly , patients with valve calcification also had a significantly greater number of deaths ( log - rank test , p = 0.001 ) . figure 3 shows the kaplan meier analysis of cardiovascular mortality ( log - rank test , p = 0.049 in aac and meier analysis of all - cause mortality ( p = 0.002 and p = 0.001 ) . kaplan meier analysis of cardiovascular mortality ( p = 0.049 and p < 0.001 ) . multivariate cox proportional hazards analyses were performed to identify factors associated with mortality . in multivariate analyses , factors that showed p univariate cox proportional hazards analysis for aac , cvc , and mortality are shown in figures 4 and 5 . the presence of aac was a significant factor associated with all - cause mortality ( hazard ratio [ hr ] : 3.149 , p = 0.025 ) in addition to lower albumin level and lower 25(oh)d level . the presence of cvc was a significant factor associated with cardiovascular mortality ( hr : 3.800 , p = 0.029 ) in addition to lower albumin level and lower 25(oh)d level . univariate cox proportional hazards analysis for aortic artery calcification , cardiac valve calcification , and all - cause mortality . univariate cox proportional hazards analysis for aortic artery calcification , cardiac valve calcification , and cardiovascular mortality . univariate and multivariate cox proportional hazards analysis for all - cause mortality bmi : body mass index ; tp : total protein ; alb : albumin ; bun : blood urea nitrogen ; scr : serum creatinine ; ua : uric acid ; ipth : intact parathyroid hormone ; 25(oh)d : 25-hydroxy vitamin d ; tg : triglyceride ; tc : total cholesterol ; hdl : high density lipoprotein ; ldl : low density lipoprotein ; fgf23 : fibroblast growth factor 23 ; p : phosphate ; ca : calcium ; cvc : cardiac valve calcification ; aac : aortic artery calcification ; hr : hazard ratio ; ci : confidence interval . univariate and multivariate cox proportional hazards analysis for cardiovascular mortality bmi : body mass index ; tp : total protein ; alb : albumin ; bun : blood urea nitrogen ; scr : serum creatinine ; ua : uric acid ; pth : parathyroid hormone ; 25(oh)d : 25-hydroxy vitamin d ; tg : triglyceride ; tc : total cholesterol ; hdl : high density lipoprotein ; ldl : low density lipoprotein ; fgf23 : fibroblast growth factor 23 ; p : phosphate ; ca : calcium ; cvc : cardiac valve calcification ; aac : aortic artery calcification ; hr : hazard ratio ; ci : confidence interval . twenty - eight ( 25.5% ) of 110 mhd patients had cvc from echocardiography , 25 ( 22.7% ) with aortic valve calcification , 10 ( 9.1% ) with mitral valve calcification , and only 1 ( 0.9% ) with tricuspid valve calcification . sixteen ( 25.0% ) of male and 12 ( 26.1% ) of female patients have cvc in our study . there is no gender difference in the incidence of cvc ( p = 0.897 ) . sixty - eight ( 61.8% ) of 110 mhd patients had visible calcification of aorta from lateral lumbar x - ray plain radiography , and the mean involved segments were 1.59 with mean aacs 4.21 0.51 scores . in analysis of the incidence of each segment of aac , thirty - eight ( 59.4% ) of male and 30 ( 65.2% ) of female have aac . there is no gender difference in the incidence of aac ( p = 0.534 ) . after 42 months follow - up , 25 ( 22.7% ) patients died , including 16 cases from cardiovascular events , 6 respiratory failure , 2 abandon treatment , and 1 deep venous thrombosis of lower extremity . in our study , 19 ( 29.7% ) male patients and 6 ( 13.0% ) female patients died during follow - up . kaplan meier analyses were performed to examine the univariate association between the presence of abdominal aortic calcification , cvc , and outcome . figure 2 shows the relationship among aac , cvc , and death from all - causes mortality and cardiovascular mortality . patients with aac had a significantly greater number of deaths from all - cause than those without aac ( log - rank test , p = 0.002 ) . similarly , patients with valve calcification also had a significantly greater number of deaths ( log - rank test , p = 0.001 ) . figure 3 shows the kaplan meier analysis of cardiovascular mortality ( log - rank test , p = 0.049 in aac and p < 0.001 in cvc ) . meier analysis of all - cause mortality ( p = 0.002 and p = 0.001 ) . kaplan meier analysis of cardiovascular mortality ( p = 0.049 and p < 0.001 ) . multivariate cox proportional hazards analyses were performed to identify factors associated with mortality . in multivariate analyses , factors that showed p < 0.05 on univariate analyses were entered as possible factors associated with mortality . univariate cox proportional hazards analysis for aac , cvc , and mortality are shown in figures 4 and 5 . the presence of aac was a significant factor associated with all - cause mortality ( hazard ratio [ hr ] : 3.149 , p = 0.025 ) in addition to lower albumin level and lower 25(oh)d level . the presence of cvc was a significant factor associated with cardiovascular mortality ( hr : 3.800 , p = 0.029 ) in addition to lower albumin level and lower 25(oh)d level . univariate cox proportional hazards analysis for aortic artery calcification , cardiac valve calcification , and all - cause mortality . univariate cox proportional hazards analysis for aortic artery calcification , cardiac valve calcification , and cardiovascular mortality . univariate and multivariate cox proportional hazards analysis for all - cause mortality bmi : body mass index ; tp : total protein ; alb : albumin ; bun : blood urea nitrogen ; scr : serum creatinine ; ua : uric acid ; ipth : intact parathyroid hormone ; 25(oh)d : 25-hydroxy vitamin d ; tg : triglyceride ; tc : total cholesterol ; hdl : high density lipoprotein ; ldl : low density lipoprotein ; fgf23 : fibroblast growth factor 23 ; p : phosphate ; ca : calcium ; cvc : cardiac valve calcification ; aac : aortic artery calcification ; hr : hazard ratio ; ci : confidence interval . univariate and multivariate cox proportional hazards analysis for cardiovascular mortality bmi : body mass index ; tp : total protein ; alb : albumin ; bun : blood urea nitrogen ; scr : serum creatinine ; ua : uric acid ; pth : parathyroid hormone ; 25(oh)d : 25-hydroxy vitamin d ; tg : triglyceride ; tc : total cholesterol ; hdl : high density lipoprotein ; ldl : low density lipoprotein ; fgf23 : fibroblast growth factor 23 ; p : phosphate ; ca : calcium ; cvc : cardiac valve calcification ; aac : aortic artery calcification ; hr : hazard ratio ; ci : confidence interval . the goal of our study is to investigate the relationship among aac , cvc and the mortality in chinese mhd patients , and which would be the better method to predict the outcome . now different vascular calcification scores have been evaluated in dialysis patient by various methods , such as x - ray plain radiography , b - mode ultrasound , and ct . the use of ct for diagnosis of aac is highly reliable and sensitive , but expensive and delivering a substantial dose of radiation . lateral lumbar x - ray is a simple method for detecting aac with cheap price , available device , and low radiation . aac score is a convenient score for clinic doctors to evaluate the severity of calcification and associated with mortality . about 94.4% of the mhd patients in australia , 56.5% in japan , and 81.0% in europe had visible aac . our previous study showed that the prevalence of aac was 60.83% in chinese mhd patients . in general population aortic arch calcification could also predict cardiovascular and all - cause mortality in mhd patients . komatsu et al . found that aortic arch calcification is linked to an increased risk of cad and is associated with cardiovascular risk factors such as age , hypertension , dyslipidemia , and dm in general population . the incidence of cvc in dialysis patients is also high , and the incidence of avc is higher than the mvc , which is similar to the result of our study . cvc is associated with the presence and severity of cad in predialysis ckd . in dialysis patients , cvc is related to mortality and age ; calcium - phosphorus product and hypoprealbuminemia are independent risk factors for cvc . the post - hoc analysis of advance study showed that cvc is a predictor of cac progression , and of greater cardiovascular vulnerability . in our study , we analyzed the association among aac , cvc , and mortality . in kaplan meier analyses and univariate analyses , both aac and cvc could predict all - cause and cardiovascular mortality in mhd patients . however , in cox proportional hazards models , only aac was a significant factor associated with all - cause mortality and cvc was a significant factor associated with cardiovascular mortality . this may be because the semi - quantitative method for evaluating aac and small size of our study . in spite of this , the results of the present study still can support the additional value of lateral lumbar x - ray films and echocardiography in mhd patients to predict their short - term outcome . it is well - known that the incidence of cardiovascular disease is relative to gender . allison et al . performed whole - body ebct scans on 650 asymptomatic subjects and found that male had higher percentage of calcification than female . although there is no gender difference in the incidence of aav and cvc in our study , male dialysis patients had a higher mortality than female . fgf23 is a novel bone - derived phosphaturic factors involved in mineral metabolism disorder and increased with the decreased kidney function and phosphate accumulates . it promotes renal phosphorus wasting and inhibits conversion of 25(oh)d to the active 1,25-dihydroxy vitamin d form . many studies showed that fgf23 has been linked to cardiovascular disease such as left ventricle hypertrophy and vascular calcification in ckd . our previous study also found that fgf23 is associated with the presence of aac in mhd patients . not just in ckd patients , a french cohort study , including 1130 healthy males , also shows that circulating fgf23 is associated with severe aac independent of other traditional risk factors . however , we did not find this relationship between fgf23 and all - cause mortality . this may be caused by the method of qualitative measurement of aortic calcification in the present study , and this study is only short - term follow - up ( 42 months ) . we need to continue to follow - up mhd patients to investigate fgf23 and long - term outcome . in ckd patients , hyperphosphatemia and calcium load the treatment of hyperphosphatemia with phosphate binder would give beneficial effect on vascular calcification progression . in our study , we did not find that phosphate and calcium are related to all - cause mortality . in cox analyses , albumin level is a protective factor for aac . huang et al . found that c - reactive protein is a predictor of aac . other inflammation factor , such as mcp-1 , was also related to all - cause and cardiovascular mortality in peritoneal dialysis patients . s100a12 , the pro - inflammatory rage - ligand , elevated in ckd 5 stage patients and is an independent predictor of mortality risk . hence , in our study , patients with low albumin level may have severe inflammation reaction and may have a link to aac . one of the important findings of this study is the association between 25(oh)d level and short - term outcome . we found that 25(oh)d is an independent predictor of cardiovascular and all - cause mortality . vitamin d deficiency has been linked to cardiovascular disease and early mortality in patients on hemodialysis , and the same association exists at earlier stages of ckd . found that plasma 25(oh)d predicted both time to death and esrd . in some studies , low doses and more physiology doses of active vitamin d previous studies demonstrated that alfa - calcidol therapy was associated with a significantly lower risk of cardiovascular and all - cause mortality in chronic hd patients . shoji et al . reported that mhd patients treated with alfa - calcidol were at reduced risk of cardiovascular death . reported that a mean daily oral vitamin d dose below 0.25 g can reduce the mortality rate by 53% in mhd patients whose serum pth levels were below 150 pg / ml . all these findings suggest that low 25(oh)d level and vitamin d3 therapy may improve cardiovascular and all - cause mortality . second , the population size in this study was small . in the future , a larger multiple - center , prospective study should be done to analyze the value of aac in dialysis patients . in conclusion , lateral lumbar x - ray plain radiography and echocardiography are simple methods to detect aac and cvc in dialysis patients . regular follow - up by x - ray and echocardiography could be useful method to stratify mortality risk in mhd patients . in an era of finite resources and ever - increasing medical possibilities , every health care system faces challenges in determining which new health technologies , including medical devices , should be introduced into clinical practice.1 while technology can improve safety2 and have other benefits , it can also bring new risks35 and contribute to the increasing cost of care.6,7 ideally , health organizations should have a systematic process both to gather relevant scientific information about a technology s safety and effectiveness and to decide whether the technology is suitable for the local setting . however , several studies have concluded that the decision - making process for the adoption of new technologies could be improved at the institutional level.810 the international network of agencies for health technology assessment defines health technology assessment ( hta ) as : [ ] the systematic evaluation of properties , effects , and/or impacts of health care technology . it may address the direct , intended consequences of technologies as well as their indirect , unintended consequences . its main purpose is to inform technology - related policymaking in health care.11 hta reports from various international , national , and provincial agencies provide comprehensive , objective , evidence - informed analyses about the safety , clinical effectiveness , cost - effectiveness , and the broader impact of health technologies , including devices , drugs , and procedures . despite the exponential growth in the numbers and the types of hta reports over the past decade and their documented impact on health care,1215 it has been observed that recommendations are not put into practice as often as their envisioned potential.1618 there are several possible reasons for this . first , local decision makers may be unaware of the wealth of hta information available to them.19,20 second , external hta agencies may not be able to consider operational factors that are critical for local decision - making , such as local needs , financial impact , and the presence of local alternatives , trained personnel , or sufficient resources . third , health care organizations may lack a systematic process by which to integrate and translate context - free hta reports with context - sensitive considerations , particularly as decision making on technology adoption at the local level is a complex process , involving many disciplines and players within an organization.2123 indeed , it has been shown that a key determinant of successful hta uptake is a clear , fair , and consistent decision - making process for the approval and introduction of new health technologies.24 to address this issue , the department of surgery and surgical services in the former calgary health region ( calgary , ab , canada ) developed and implemented a systematic decision - making process to introduce new health technologies called the local hta decision support program , which comprises sets of forms and tools.25 the forms gather context - free , scientific evidence about the technology , such as hta reports , and context - sensitive information about local needs and constraints . the tools provide decision guides based on explicit criteria26 to assist an interdisciplinary advisory committee in translating the evidence to make a recommendation to the surgical executive committee , who then makes the final decision on whether and under what conditions the technology will be used . over a five - year period , 68 technology requests were reviewed using this program . as well as producing yes or no decisions on some technologies , the local hta decision support program gave other technologies restricted approval , with full approval contingent on satisfying clinical outcomes reporting , training protocol development , or funding.25 thus , the program provides a strong link between hta , outcomes measures and research , and improvement in the quality of care . we have observed considerable interest on the part of health care organizations in developing a similar program to combine the evidence of hta reports with local operational considerations to produce technology adoption decisions for their own needs . an alternative would be to adapt a preexisting program to the needs of the new setting rather than developing one de novo . the present project was launched to develop a framework and tools for adapting the local hta decision support program for use by other departments in the calgary health region . the operation of the program itself has been described in detail elsewhere.25 the purpose of this article is to describe the adaptation framework and tools , identify the key elements required for successful participation , and describe the lessons learned to support others who wish to use this framework for adapting our local hta decision support program , or a similar one , for their local needs . at the time of this project , the calgary health region was an integrated , primarily urban health authority in alberta , canada . it provided services across the continuum of care , from community health services to acute tertiary care , and served about one million people . its administrative structure comprised 14 major regional clinical departments , which worked within a portfolio structure . each portfolio was overseen by an executive director ( administrator ) and a medical director ( physician ) , who worked with the senior management team in the region . since then , the calgary health region has been restructured as part of a single provincial health services organization ( alberta health services ) . the department of surgery and surgical services in the calgary area comprises 286 surgeons in 14 divisions . following an extensive hta education initiative , there was a desire to see the local hta decision support program originally developed by the department of surgery and surgical services adopted by other departments within the calgary health region.19,27 with strong support by senior management , the present project was launched to develop a framework and tools for adapting the local hta decision support program for use by other departments in the region . adaptation of the local hta decision support program involved six steps organized into three main phases : set - up , review and adaptation , and finalization . each phase included a set of objectives , tasks , and documents ( table 1 ) modified from the adapte framework.28 the set - up phase consisted of tasks to be completed before the adaptation process . step 1 was the development of a local health technology assessment decision support program : review and adaptation manual,29 which contained a description of the local hta decision support program , hta - related reference materials for education purposes ( for example , table 2 ) , a project time line , and tips on program administration and evaluation . step 2 involved identification , education , and selection of departments ready and willing to review and adapt the program . in addition to surgery and surgical services , we approached ten other departments within the calgary health region we scheduled semi - structured interviews and meetings with each department s clinical and administrative heads and their executive committees to introduce the project , gather initial feedback , and gain approval for their participation in the adaptation process . each department s readiness for the project was determined using an assessment of readiness tool ( table 3 ) . outcomes required to proceed included the department s desire to change its manner of introducing new health technologies and a commitment of resources and personnel , namely the appointment of the local hta leaders ( a physician and administrator team ) to oversee the review and adaptation phase . we then met with these leaders to ensure they understood the local hta decision support program , the material in the review and adaptation manual,29 and what would be required for the review and adaptation process . the review and adaptation phase involved independent and joint reviews of the source program by participating departments . for step 3 , each department s appointed local hta leaders were charged with conducting an independent review with their membership either in principle by evaluating the structure , forms , and processes of the program or in practice by evaluating actual requests for new technologies from their members . each participating department was asked to provide written feedback using the points to consider questionnaire ( table 4 ) . step 4 consisted of a joint review by all participating departments in the form of full - day retreats . the retreat objectives were to learn from each department s experience in reviewing the program , to share input and ideas for improvement , to explore the need for consistency and the need for flexibility , and to determine future directions and next steps . retreats were divided into five sessions that followed the outline of the points to consider questionnaire ( table 4 ) to systematically review the program s policy , flow , structure , content , and funding . participants were asked to share their observations and to make joint recommendations to improve the program . retreat reports were produced that incorporated structured feedback from participants during working sessions and notes made by the facilitator and research team members . the finalization phase involved revision of the local hta decision support program and review of the adaptation process itself . for step 5 , feedback was compiled from the semi - structured interviews and meetings held with each of the local hta leaders from individual departments , written comments provided on the points to consider questionnaire reporting form , and the retreat reports . data were analyzed using a content analysis - type approach.30 major themes and subthemes were developed through comparison and categorization of information gathered . the team met to discuss the outcomes and reached consensus in cases where discrepancies arose . the outcomes were then interpreted into meaningful concepts pertaining to the local hta decision support program , and the program was revised accordingly . for step 6 , we developed a project evaluation questionnaire and gave it to all participants at the end of each retreat to evaluate the review and adaptation process used in this project . at the time of this project , the calgary health region was an integrated , primarily urban health authority in alberta , canada . it provided services across the continuum of care , from community health services to acute tertiary care , and served about one million people . its administrative structure comprised 14 major regional clinical departments , which worked within a portfolio structure . each portfolio was overseen by an executive director ( administrator ) and a medical director ( physician ) , who worked with the senior management team in the region . since then , the calgary health region has been restructured as part of a single provincial health services organization ( alberta health services ) . the department of surgery and surgical services in the calgary area comprises 286 surgeons in 14 divisions . following an extensive hta education initiative , there was a desire to see the local hta decision support program originally developed by the department of surgery and surgical services adopted by other departments within the calgary health region.19,27 with strong support by senior management , the present project was launched to develop a framework and tools for adapting the local hta decision support program for use by other departments in the region . adaptation of the local hta decision support program involved six steps organized into three main phases : set - up , review and adaptation , and finalization . each phase included a set of objectives , tasks , and documents ( table 1 ) modified from the adapte framework.28 the set - up phase consisted of tasks to be completed before the adaptation process . step 1 was the development of a local health technology assessment decision support program : review and adaptation manual,29 which contained a description of the local hta decision support program , hta - related reference materials for education purposes ( for example , table 2 ) , a project time line , and tips on program administration and evaluation . step 2 involved identification , education , and selection of departments ready and willing to review and adapt the program . in addition to surgery and surgical services , we approached ten other departments within the calgary health region we scheduled semi - structured interviews and meetings with each department s clinical and administrative heads and their executive committees to introduce the project , gather initial feedback , and gain approval for their participation in the adaptation process . each department s readiness for the project was determined using an assessment of readiness tool ( table 3 ) . outcomes required to proceed included the department s desire to change its manner of introducing new health technologies and a commitment of resources and personnel , namely the appointment of the local hta leaders ( a physician and administrator team ) to oversee the review and adaptation phase . we then met with these leaders to ensure they understood the local hta decision support program , the material in the review and adaptation manual,29 and what would be required for the review and adaptation process . the review and adaptation phase involved independent and joint reviews of the source program by participating departments . for step 3 , each department s appointed local hta leaders were charged with conducting an independent review with their membership either in principle by evaluating the structure , forms , and processes of the program or in practice by evaluating actual requests for new technologies from their members . each participating department was asked to provide written feedback using the points to consider questionnaire ( table 4 ) . step 4 consisted of a joint review by all participating departments in the form of full - day retreats . the retreat objectives were to learn from each department s experience in reviewing the program , to share input and ideas for improvement , to explore the need for consistency and the need for flexibility , and to determine future directions and next steps . retreats were divided into five sessions that followed the outline of the points to consider questionnaire ( table 4 ) to systematically review the program s policy , flow , structure , content , and funding . participants were asked to share their observations and to make joint recommendations to improve the program . retreat reports were produced that incorporated structured feedback from participants during working sessions and notes made by the facilitator and research team members . the finalization phase involved revision of the local hta decision support program and review of the adaptation process itself . for step 5 , feedback was compiled from the semi - structured interviews and meetings held with each of the local hta leaders from individual departments , written comments provided on the points to consider questionnaire reporting form , and the retreat reports . data were analyzed using a content analysis - type approach.30 major themes and subthemes were developed through comparison and categorization of information gathered . the team met to discuss the outcomes and reached consensus in cases where discrepancies arose . the outcomes were then interpreted into meaningful concepts pertaining to the local hta decision support program , and the program was revised accordingly . for step 6 , we developed a project evaluation questionnaire and gave it to all participants at the end of each retreat to evaluate the review and adaptation process used in this project . comments provided described how some technologies were introduced , removed , or replaced with alternatives without adequate evidence review or input from clinicians . to give some examples , however , the upgraded device was incompatible with existing hand pieces and added a large generator to an already tight space . although hta reports indicated good evidence for efficacy , the device failed to improve patient outcomes in our local setting , due to the lack of lead time to allow for appropriate training . however , no funds were given for its high operating cost , and this unbudgeted expense then fell to the health care system . new endoscopes were purchased , but they required specialized sterilization equipment , which was not available in all operating rooms . numerous examples were also given of requests for technologies that were based only on information obtained from the manufacturers representatives . consequently , department administrators and physician leaders wanted a system that would provide greater accountability and broader review for technology adoption . there was also some resistance to change particularly around the perceived time and work required to run a local hta decision support program . in addition to the department of surgery and surgical services , we were successful in recruiting eight out of the ten departments originally approached for a total of nine departments : six clinical departments and three administrative departments . all the clinical departments appointed a physician and an administrator as local hta coleaders , while the administrative departments appointed only an administrator as their hta leader . the department of surgery and surgical services already had a functioning physician - administrator team as the hta coleaders . of the two departments that declined to participate in the project , one was in the midst of organizational transition after hiring a new department head and the other was not interested in developing its own program as its technology purchasing was overseen by the department of surgery and surgical services . four departments reviewed the local hta decision support program in practice ( three invited departments as well as surgery and surgical services ) , and five departments reviewed the program in principle . all participating departments submitted their individual review feedback questionnaire and participated in the joint retreats . in total , we met with 67 individuals during the semi - structured interviews and meetings with each department s clinical and administrative heads and their executive committees , including physicians , program managers , nurse clinicians , patient care managers , health services directors , health services managers , clinical product specialists , clinical safety leaders , researchers , and financial analysts , as well as the senior administrators . thirteen individuals participated in the retreat for the first review cycle ; 16 participated in the retreat for the second review cycle . during the joint retreats , a significant amount of time what section of the points to consider questionnaire ( table 4 ) ; that is , what information needs to be gathered to ensure that the department s local hta committee would have enough information to decide if a technology could be simply approved or if it needed further assessment ? this was a contentious issue , because it is difficult to strike a balance between avoiding unnecessary work while still being able to protect patient safety and reduce risk when introducing new technologies . there was good consensus on the why section of the questionnaire ; that is , the need for a systematic and transparent decision support program for technology review and adoption and a clear policy statement . there was also good consensus on the how , who , and funding sections of the questionnaire , with the general recommendation that the details of program operation should be left to the discretion of individual departments . similar comments were brought forward by those who reviewed the program in practice as compared with those who reviewed in principle . to evaluate the review and adaptation process used in this project , the research team reviewed the responses to the project evaluation questionnaire that was given to participants after each of the two retreats . for the may 2007 retreat , 89%100% of respondents ( n=9 ) agreed or strongly agreed that the retreat objectives were met and 100% agreed or strongly agreed that the desired outcomes were achieved . these outcomes were the revised local hta decision - support program is adaptable to the needs of various clinical departments and i am committed to the next steps . for the november 2007 retreat , 80%100% of respondents ( n=10 ) agreed or strongly agreed that the retreat objectives were met , and 90%100% agreed or strongly agreed that the desired outcomes were achieved . these outcomes were : the revised local hta decision support program is adequate to go live for january 2008 implementation , and i am committed to continued participation . one person disagreed with the first outcome statement , but no other participants scored disagree or strongly disagree for any of the other objective or outcome statements . open - ended comments on the project evaluation questionnaire revealed an overwhelming desire to continue face - to - face meetings . participants found value in hearing the experiences of others using or reviewing the program , gathering feedback from different perspectives , and working collaboratively to come to consensus . as well , participants affirmed the value in having a fair , standardized , and consistent process for technology adoption across the various specialties within the calgary health region . using the above described methodology , two cycles of the review and adaptation and finalization phases were performed with invited departments . as part of another project,26 an additional cycle of review comments provided described how some technologies were introduced , removed , or replaced with alternatives without adequate evidence review or input from clinicians . to give some examples , however , the upgraded device was incompatible with existing hand pieces and added a large generator to an already tight space . although hta reports indicated good evidence for efficacy , the device failed to improve patient outcomes in our local setting , due to the lack of lead time to allow for appropriate training . however , no funds were given for its high operating cost , and this unbudgeted expense then fell to the health care system . new endoscopes were purchased , but they required specialized sterilization equipment , which was not available in all operating rooms . numerous examples were also given of requests for technologies that were based only on information obtained from the manufacturers representatives . consequently , department administrators and physician leaders wanted a system that would provide greater accountability and broader review for technology adoption . there was also some resistance to change particularly around the perceived time and work required to run a local hta decision support program . in addition to the department of surgery and surgical services , we were successful in recruiting eight out of the ten departments originally approached for a total of nine departments : six clinical departments and three administrative departments . all the clinical departments appointed a physician and an administrator as local hta coleaders , while the administrative departments appointed only an administrator as their hta leader . the department of surgery and surgical services already had a functioning physician - administrator team as the hta coleaders . of the two departments that declined to participate in the project , one was in the midst of organizational transition after hiring a new department head and the other was not interested in developing its own program as its technology purchasing was overseen by the department of surgery and surgical services . four departments reviewed the local hta decision support program in practice ( three invited departments as well as surgery and surgical services ) , and five departments reviewed the program in principle . all participating departments submitted their individual review feedback questionnaire and participated in the joint retreats . in total , we met with 67 individuals during the semi - structured interviews and meetings with each department s clinical and administrative heads and their executive committees , including physicians , program managers , nurse clinicians , patient care managers , health services directors , health services managers , clinical product specialists , clinical safety leaders , researchers , and financial analysts , as well as the senior administrators . thirteen individuals participated in the retreat for the first review cycle ; 16 participated in the retreat for the second review cycle . during the joint retreats , a significant amount of time what section of the points to consider questionnaire ( table 4 ) ; that is , what information needs to be gathered to ensure that the department s local hta committee would have enough information to decide if a technology could be simply approved or if it needed further assessment ? this was a contentious issue , because it is difficult to strike a balance between avoiding unnecessary work while still being able to protect patient safety and reduce risk when introducing new technologies . there was good consensus on the why section of the questionnaire ; that is , the need for a systematic and transparent decision support program for technology review and adoption and a clear policy statement . there was also good consensus on the how , who , and funding sections of the questionnaire , with the general recommendation that the details of program operation should be left to the discretion of individual departments . similar comments were brought forward by those who reviewed the program in practice as compared with those who reviewed in principle . to evaluate the review and adaptation process used in this project , the research team reviewed the responses to the project evaluation questionnaire that was given to participants after each of the two retreats . for the may 2007 retreat , 89%100% of respondents ( n=9 ) agreed or strongly agreed that the retreat objectives were met and 100% agreed or strongly agreed that the desired outcomes were achieved . these outcomes were the revised local hta decision - support program is adaptable to the needs of various clinical departments and i am committed to the next steps . no participant scored disagree or strongly disagree for any of the objective or outcome statements . for the november 2007 retreat , 80%100% of respondents ( n=10 ) agreed or strongly agreed that the retreat objectives were met , and 90%100% agreed or strongly agreed that the desired outcomes were achieved . these outcomes were : the revised local hta decision support program is adequate to go live for january 2008 implementation , and i am committed to continued participation . one person disagreed with the first outcome statement , but no other participants scored disagree or strongly disagree for any of the other objective or outcome statements . open - ended comments on the project evaluation questionnaire revealed an overwhelming desire to continue face - to - face meetings . participants found value in hearing the experiences of others using or reviewing the program , gathering feedback from different perspectives , and working collaboratively to come to consensus . as well , participants affirmed the value in having a fair , standardized , and consistent process for technology adoption across the various specialties within the calgary health region . using the above described methodology , two cycles of the review and adaptation and finalization phases were performed with invited departments . as part of another project,26 an additional cycle of review decision making is a complex process , involving many disciplines and players within an organization when introducing new health technologies , including medical devices.23 because of cultural and organizational differences , a recommendation for technology adoption in one local setting may not be appropriate in another , even when the scientific evidence , such as that supplied by hta reports , is the same . thus , careful consideration of the specific questions relevant to local needs , priorities , legislation , policies , values , norms , as well as human and material resources , is as necessary as acquiring impartial scientific evidence . the local hta decision support program developed initially by the department of surgery and surgical services of the calgary health region is one attempt to provide a model ( structure , processes , criteria , and decision tools ) for decision making that systematically gathers scientific , operational , and value - based information for consideration when introducing new health technologies . similar programs exist elsewhere.3134 any program that attempts to integrate context - free scientific evidence about a technology with context - sensitive information about the setting in which it will be used must be appropriate and relevant for each local organizational body . however , small rural and community hospitals or health authorities with fewer financial , personnel , and community resources may be poorly positioned for the investment required for de novo decision support program development . programs developed by large health care systems can be adapted and applied successfully to rural settings , provided that needed tools are provided for the adaptation process.35 this paper provides an adaptation process that can be used in lieu of de novo program development , much like processes for adapting clinical practice guidelines.36,37 the adaptation process described and used in this article involved six steps organized into three main phases : the set - up phase involved the development of a manual and identifying and educating interested departments , the review and adaptation phase involved assisting the departments to conduct an actual review and adaptation of the program , and the finalization phase involved program revision and evaluation of the adaptation process . during the set - up phase , we found a general dissatisfaction of each department with its traditional decision - making processes when introducing new health technologies . similar dissatisfaction has been observed by others10 and provides a good opportunity to restructure these processes . to do this , we found that provision of a clearly written document , the local health technology assessment decision support program : review and adaptation manual , was vital to educate members about the value of hta and a structured decision support program and to capture interest and buy - in . hta , therefore having well - written program documents and educational reference materials was vital to ensure their learning and understanding . a key document proved to be the table that explained the distinction between hta producers and hta users . we found that understanding this difference was important for understanding the rationale for a local hta decision support program . the program does not duplicate the efforts of hta producers ; it complements their work by acting as the receptor to use hta reports . the assessment of readiness tool was a critical element of the set - up phase . it required that participating departments demonstrate readiness for change and appoint local hta physician and administrative leaders . during the review and adaptation phase , the research team provided ongoing assistance to participating departments by means of phone , email , and face - to - face meetings and education . we ensured that we provided a variety of methods to solicit feedback , as recommended by others.38 we found that the points to consider questionnaire ( why , how , who , what , funding ) was essential to ensure that all important program review questions would be systematically discussed by reviewing departments . the request that each department appoint an hta physician leader and an hta administrative leader was crucial to the success of the project . while physicians were more focused on clinical evidence , training and credentialing issues , and other such clinically relevant topics , their administrative counterparts ensured that infrastructure implications , cost , and organizational impact were reviewed . similarly , the presence of relevant stakeholders , such as physicians , program managers , nurse clinicians , patient care managers , health services directors , health services managers , clinical product specialists , clinical safety leaders , researchers , financial analysts , and high - level health services administrators in the review and adaptation process can provide critical advantages . first , it ensures that those most likely to use the program have an opportunity to offer feedback and to identify problems before it is finalized . second , it gives administrators the opportunity to consider the impact on the organization of implementing the program and to begin preparing for its future adoption . third , the solicitation of practitioner feedback serves as the first wave of dissemination of the proposed program . fourth , experience elsewhere has shown that when surgeons guide the appraisal of new and emerging surgical technologies , the benefits of an evidence - informed approach in the provision of high - quality patient care are realized.39 the recommendation taken forward to the finalization phase was that the content ( what ) should be standardized across departments and an interdisciplinary team should review the application , but that administrative details ( who and how ) should be left to the discretion of each department . this reflects the importance participants saw in having a fair , standardized , and consistent decision support program for technology adoption across specialties within the institution while acknowledging flexibility of operation . participants demonstrated a strong desire to adopt the decision support program and to keep the review process going by engaging in regular face - to - face group collaboration and interaction . in fact , the review and adaptation and finalization phases can be repeated on a regular scheduled basis in a retreat setting , resulting in a continuous improvement cycle , as well as developing a community of practice , which is known to be effective in sustaining the viability and improvement of programs of interest.40 we identified several obstacles to long - term implementation of the program . first , staff turnover during the course of implementation can result in a continual need to educate and update decision makers . second , there is yet no process by which any cost savings realized by the adoption of new technologies can be attributed back to the operation of the program . third , shifting priorities of a health care system ( for example , funds diverted to pandemic flu inoculations ) may result in the cancellation or delay in the implementation of a recommended new technology . these obstacles are in addition to challenges encountered during the project itself , including finding common meeting times among all stakeholders , some resistance to change ( ie , the perceived fear that running a decision support program when introducing new health technologies may be labor intensive ) , and finding funding for the appointment of hta leaders and support personnel . benefits of the project included increased research - mindedness among clinicians , staff , and administrators who participated in the decision support program review and adaptation process , increased awareness of hta , increased understanding and appreciation of clinicians and administrators in the complexity of evidence - informed technology introduction process , and reduced barriers to implementation of the proposed program as a result of buy - in from the participating stakeholders . these observations will need to be confirmed as others use the decision - making program review and adaptation framework . in summary , we present a three - phase framework for reviewing and adapting a well - developed local hta decision support program for application in different settings . this adaptation process divides a complicated process into a discrete step - by - step approach , which can be repeated regularly to improve quality , and ensures that the adaptation process is systematic , rigorous , and interdisciplinary . we conclude that the adaptation of a preexisting program may reduce duplication of effort , save resources , raise health care providers awareness of hta , and foster constructive stakeholder engagement , which enhances the legitimacy of evidence - informed recommendations for introducing new health technologies . we encourage others to use this framework for decision support program adaptation and report their experiences .

background : this study was to investigate the relationship among aortic artery calcification ( aac ) , cardiac valve calcification ( cvc ) , and mortality in maintenance hemodialysis ( mhd ) patients.methods:all mhd patients in shanghai ruijin hospital in july 2011 were included . to follow up for 42 months , clinical data , predialysis blood tests , echocardiography , and lateral lumbar x - ray plain radiography results were collected . plasma fgf23 level was measured using a c - terminal assay.results:totally , 110 mhd patients were involved in this study . of which , 64 ( 58.2% ) patients were male , the mean age was 55.2 1.4 years old , and the median dialysis duration was 29.85 ( 3.0225.5 ) months . about 25.5% of the 110 mhd patients had cvc from echocardiography while 61.8% of the patients had visible calcification of aorta from lateral lumbar x - ray plain radiography . after 42 months follow - up , 25 ( 22.7% ) patients died . kaplan meier analysis showed that patients with aac or cvc had a significant greater number of all - cause and cardiovascular deaths than those without . in multivariate analyses , the presence of aac was a significant factor associated with all - cause mortality ( hazard ratio [ hr ] : 3.149 , p = 0.025 ) in addition to lower albumin level and lower 25-hydroxy vitamin d ( 25(oh)d ) level . the presence of cvc was a significant factor associated with cardiovascular mortality ( hr : 3.800 , p = 0.029 ) in addition to lower albumin level and lower 25(oh)d level.conclusion:lateral lumbar x - ray plain radiography and echocardiography are simple methods to detect aac and cvc in dialysis patients . the presence of aac and cvc was independently associated with mortality in mhd patients . regular follow - up by x - ray and echocardiography could be a useful method to stratify mortality risk in mhd patients . purposeintroducing new health technologies , including medical devices , into a local setting in a safe , effective , and transparent manner is a complex process , involving many disciplines and players within an organization . decision making should be systematic , consistent , and transparent . it should involve translating and integrating scientific evidence , such as health technology assessment ( hta ) reports , with context - sensitive evidence to develop recommendations on whether and under what conditions a new technology will be introduced . however , the development of a program to support such decision making can require considerable time and resources . an alternative is to adapt a preexisting program to the new setting.materials and methodswe describe a framework for adapting the local hta decision support program , originally developed by the department of surgery and surgical services ( calgary , ab , canada ) , for use by other departments . the framework consists of six steps : 1 ) development of a program review and adaptation manual , 2 ) education and readiness assessment of interested departments , 3 ) evaluation of the program by individual departments , 4 ) joint evaluation via retreats , 5 ) synthesis of feedback and program revision , and 6 ) evaluation of the adaptation process.resultsnine departments revised the local hta decision support program and expressed strong satisfaction with the adaptation process . key elements for success were identified.conclusionadaptation of a preexisting program may reduce duplication of effort , save resources , raise the health care providers awareness of hta , and foster constructive stakeholder engagement , which enhances the legitimacy of evidence - informed recommendations for introducing new health technologies . we encourage others to use this framework for program adaptation and to report their experiences .

in anterior cruciate ligament ( acl ) reconstruction , the use of bone patellar tendon bone graft ( bptb ) has been decreasing due to increased anterior knee pain , weakness of extensor power and difficulty of kneeling position . instead , the use of hamstring tendon has been increasing1,2 ) ; however , the disadvantages of hamstring grafts include weaker fixation strength compared to bptb grafts and 24-week period for complete bone - to - tendon healing3 ) . adequate fixation has been considered essential for good outcomes of acl reconstruction and a variety of graft fixation methods have been introduced . the compression method allows for early firm fixation and healing with tight bone - tendon interface and enables close fixation to the acl footprint , but it has low failure load and stability4,5 ) . the expansion fixation mechanism can be advantageous in obtaining secure fixation because two cross pins transversely inserted through a graft provides a centrifugal pressure on the femoral tunnel , but treatment results depend on the press - fit of the graft , bone density around the femoral tunnel , and correct placement of cross pins through the graft tendon6,7,8 ) . the suspension methods are sub - classified into cortical , cancellous and , cortio - cancellous suspension methods9 ) . the cortical suspension method provides good fixation strength , but it has a bungee cord effect10 ) and a windshield wiper effect11 ) due to the long fixation point from the articular surface . the cortico - cancellous suspension method has strong stability and stiffness due to the use of a metaphyseal crosspin . to et al.12 ) reported that stiffness of the graft fixation complex was more affected by fixation method than the graft type in a cadaver study . intercondylar cortico - cancellous fixation close to the articular surface is expected to offer better results than the endobutton fixation . speirs et al.13 ) reported that the cortico - cancellous suspensory fixation method required a short graft length due to fixation of the cross pin within the metaphysis , the lowest creep and cyclic elongation amplitude , and the highest strength and stiffness of all the tested devices . in particular , the pinn - acl crosspin ( conmed linvatec , largo , fl , usa ) system was found to be the most rigid and strongest of all the tested reconstruction systems . therefore , the cortico - cancellous fixation method seems to have the advantages of short graft , stability , and stiffness , and the pinn - acl crosspin implant features the proprietary self - reinforced poly - l - lactide acid polymer enabling it to be the strongest bioabsorbable implant . the cross pin absorption begins in vivo approximately within 15 to 24 weeks after insertion , the continuous loop is composed of high strength polyethylene fiber , and the ultimate pullout tensile strength is 1700n14 ) ( fig . , we analyzed the mid - term results of acl reconstruction using the pinn - acl crosspin system , a cortico - cancellous suspension method device . we hypothesized that acl reconstruction using the pinn - acl crosspin system would significantly improve manual stability , anterior - posterior laxity measured by instrument , and functional score . from june 2007 to july 2008 , 43 of 46 patients with ruptured acls were evaluated . the patients underwent single - bundle acl reconstruction using a 4-strand semitendinosus tendon ( semi - t ) with the pinn - acl crosspin system . three patients were excluded due to conditions that might affect the results : articular cartilage damage and osteoarthritis in 1 patient , subtotal meniscectomy in 1 patient , and total meniscectomy in 1 patient . all included patients were male with a mean age of 28.7 years ( range , 18 to 54 years ) . the acl reconstruction was performed on the right knee in 24 patients and on the left knee in 19 patients . the mean follow - up period was 50 months ( range , 48 to 61 months ) . associated lesions were meniscal tear in 15 patients , which required meniscus repair in 5 and partial meniscectomy in 10 , and medial collateral ligament injury in 2 patients , which was treated by conservative methods . a 4-strand semi - t was used as a graft . a longer than 28 cm semi - t was harvested with an additional 2 cm of periosteum extension and was folded twice to be a 4-strand graft15 ) . the mean length of the graft was 7.2 cm and the diameter was 8.2 cm ( fig . depending on the diameter of the femoral tunnel , a positioning rod ( 8 mm or 9 mm ) of appropriate size was selected and assembled onto the u - guide . when the u - guide assembly was fully inserted , the laser etch marks on the positioning rod indicated the length of the femoral tunnel . after the u - guide was fully inserted into the tunnel , the u - guide body was rotated until the transverse cannula mounted on the u - guide body was directed toward the lateral condyle . each cross pin had a cortical length designed to occupy the cortical side of the transverse tunnel . a cross pin of proper length was determined as the one whose cortical length was less than , or equal to , the measured cortical tunnel length . after selecting the proper size cross pin , the hamstring graft construct ( i.e. , graft harness and graft bundle assembly ) was drawn into the knee using the graft passing guide pin . the lead suture was passed on the graft harness through the eyelet of the graft passing guide pin and , while maintaining lateral to medial alignment of the axis of the eyelet in the graft harness , the graft construct was passed into the tibial tunnel . it was firmly pulled on the graft construct until fullly seated in the femoral socket . a sheathed scope could be placed into the transverse cannula to visualize the alignment of the axis of the graft harness eyelet with the axis of the transverse tunnel . the crosspin driver was inserted into the proximal end of the implant and tapped with a mallet to move the implant through the transverse cannula and into the transverse tunnels . the corsspin implant , pressed into the lateral transverse tunnel , was advanced with the driver and a mallet until it stops while pulling out the transverse cannula . the position of the femoral tunnel was at the center of the footprint , directed 10:30 ( or 1:30 ) o'clock position . 3 ) . we tried to preserve the remnants of acl as much as possible . tibial side fixation was done using a bioabsorbable screw and then post - tied with a washer and screw . range of motion exercises were allowed at 2 weeks after surgery with extension locking braces applied . up to 90 of active range of motion was permitted for 4 weeks and full range of motion exercise was performed thereafter . from 6 weeks after surgery , patients followed a usual rehabilitation program17 ) . in patients who had undergone concomitant meniscal repair , anterior instability was evaluated on the day of admission using the lachman test and the kneelax3 arthrometer ( monitored rehab systems , haarlem , netherlands)18 ) . rotational instability was evaluated using the pivot - shift test with the patient under anesthesia immediately before surgery . functional knee score was evaluated using the international knee documentation committee ( ikdc ) subjective score and objective grade . student 's t - test was used to analyze parametric continuous data and chi - square test was used for non - parametric data . statistical significance was accepted for p - values of < 0.05 , and spss ver . 21.0 ( ibm corp . the range of motion was improved to normal without limitation at final follow - up . the lachman test results were improved form grade ii ( n=40 ) or iii ( n=3 ) to grade i ( n=3 ) or grade 0 ( n=40 ) at final follow - up ( p=0.001 ) . the pivot - shift test results were improved from grade i ( n=20 ) or ii ( n=10 ) to grade i ( n=8 ) or grade 0 ( n=22 ) at final follow - up ( p=0.001 ) . the ikdc subjective score was improved to 88.7 and the objective grades a and b were noted in 93% of the patients at final follow - up ( p=0.039 , 0.001 ) . anterior laxity measured by the kneelax3 arthrometer was improved from 6.74.5 mm preoperatively to 2.11.0 mm at final follow - up ( p=0.021 ) ( table 1 ) . three cases of complications occurred : a graft re - rupture was treated with revision reconstruction at 2 years after surgery ; a superficial infection on the tibial side was improved after debridement ; and a deep knee infection on the tibial side found at 2 weeks after surgery was identified as methicillin resistance staphylococcus aureus and treated with thorough arthroscopic debridement , massive irrigation , and 4 weeks of antibiotic injection . in this study , anterior instability evaluated using the lachman test and kneelax3 arthrometer , and rotational instability evaluated by the pivot - shift test were significantly improved after surgery . the functional knee score evaluated using the ikdc score & grade was also remarkably improved after surgery . seo et al.19 ) reported on 56 cases of acl reconstruction using the pinn - acl crosspin system . in the study , the side - to - side difference measured by the kt-1000 arthrometer was 2.4 mm at a mean of 14.5-month follow - up and the mean ikdc score was 87.3 . kong et al.20 ) reported on 56 cases of acl reconstruction using rigidfix , another crosspin system . in their study , the mean side - to - side difference was 2.1 mm and 98.2% of the cases had ikdc grades a or b. streich et al.21 ) reported about 25 cases of single bundle acl reconstruction with a single semi - t : the mean side - to - side difference was 0.94 mm , the pivot - shift test was grade 0 in 19 cases , more than grade 1 in 6 cases , and the mean ikdc score was 88.6 . seo et al.19 ) reported that the incidence of crosspin - femoral tunnel mismatch was high . to prevent this problem , they tried to firmly fix the drill guide sheath to the femur or create a short femoral tunnel to perform drilling at almost perpendicular direction to the cortical bone , but this technique requires further improvement of tools for minimization of complications . in our study , we encountered a mismatch between the harness hole within the femoral tunnel and the crosspin tunnel caused by rotation of the harness in bone tunnel . we solved this problem by firmly fixing the guide assembly and switching from a small sized stick to a larger one of the same size of the harness hole to make 90% of the harness hole coincide with the crosspin tunnel . yamazaki et al.22 ) and zantop et al.23 ) reported on the optimal length of the soft - tissue graft within a bone tunnel . they concluded that a graft length of over 15 mm does not influence the kinematic or structural properties of the knee joint . although lipscomb et al.24 ) indicated that slight or no deficits were observed in the knee flexor strength , most reports suggested that deficits of 10% to 20% in the knee flexor strength are common after acl reconstruction using both semi - t and gracilis tendon autograft25,26 ) . the single 4-strand semi - t tendon acl graft was shorter but could be made thicker , had biomechanical benefits , and decreased donor site morbidity by not harvesting the gracilis tendon27 ) . for a four - strand hamstring graft , at least a graft length of 7 cm is recommended15 ) ( fig . , the minimum required semi - t tendon length is 28 cm for a four - strand semi - t graft . it is possible to obtain an additional 2 cm of semi - t tendon by including the periosteum15 ) . in this study the mean graft length was 7.2 cm , the mean diameter was 8.2 mm , which was thicker than the semi - t / gracilis 4-strand graft . so , we could expect less decrease of the knee flexor strength without harvesting of the gracilis tendon , enhanced tendon healing to the bone tunnel due to inclusion of the periosteum in the graft and a thicker graft28 ) . the pinn - acl crosspin instrument could be one of the useful cortio - cancellous suspensory devices for femoral fixation , allowing for easy fixation with a shorter graft ( single semi - t 4-strand ) and reducing donor site morbidity . the other crosspin , rigidfix system , requires a 3-cm long graft in the femoral bone tunnel for adequate fixation . however , the pinn - acl crosspin needs a 1.5 - 2 cm graft for adequate fixation . one case of graft re - rupture occurred , but it was not related to the fixation method . regarding the one case of deep infection and another case of superficial infection , these two infections were improved after debridement . we suspect that the cause of infection might have been contamination of the guide assembly because the pinn - acl crosspin instrument was complex and composed of several small parts and guide assembly . so , we believe there is a need for thorough cleansing and sterilization including foreign body particle removal from the guide assembly before surgery . maletis et al.29 ) evaluated the incidence of postoperative acl reconstruction infections in the total 10,626 cases and concluded that graft choice would make a difference . the overall incidence of surgical site infection ( ssi ) was 0.48% ( n=51 ) , with 17 ( 0.16% ) superficial infections and 34 ( 0.32% ) deep infections . hamstring tendon autografts ( 0.61% ) had the highest incidence of deep ssis of the total graft types ( bptb autograft 0.07% vs. allograft 0.27% ) . after adjusting for age , sex , and body mass index , the likelihood of a patient with a hamstring autograft having a deep ssi was 8.24 times higher than someone receiving a bptb autograft . van tongel et al.30 ) reported the incidence of septic arthritis after acl reconstruction using semi - t / gracilis autograft was 0.51% . the limitations of this study are no inclusion of a control group , retrospective study design , and no performance of radiologic evaluation . this study demonstrated that good results can be obtained after single - bundle acl reconstruction using 4-strand semi - t tendon with the pinn - acl crosspin system at a minimum follow - up of 48 months . we believe the pinn - acl crosspin system is a useful instrument for shorter graft fixation . autosomal - recessive renal tubular dysgenesis ( ar - rtd ) is a rare inherited disorder caused by mutations in the genes encoding any of the components of the renin angiotensin system ( ras ) including renin , angiotensinogen , angiotensin - converting enzyme ( ace ) and type 1 angiotensin ii receptor . fetuses may die in utero , and most neonates die soon after birth with persistent anuria , respiratory failure and refractory hypotension . the histopathological hallmark of the disease is the absence or incomplete development of cortical convoluted proximal tubules [ 2 , 3 ] . since this disease was first described by allanson et al . in 1983 , > 100 cases of rtd with or without genetic defects have been reported . while most previous reports have described ar - rtd as a lethal disease , five recent cases have reportedly survived the neonatal period [ 58 ] . here , we report another case of ar - rtd associated with ace mutations who survived for over 2 years . the first and second pregnancies were uneventful but the third pregnancy was complicated by anhydramnios and was terminated at 28 weeks of gestation . the patient was born at 32 weeks and 4 days of gestation due to pre - term labor . the weight was 1960 g ( 1050th percentile ) and the height was 41.5 cm ( 1050th percentile ) . the baby required assisted ventilation immediately after birth due to respiratory distress , and inotropes were started at 1 h after birth due to hypotension . the patient developed a pneumoperitoneum due to ileal perforation at 7 h after birth and he underwent emergency ileostomy . hypotension was aggravated after surgery and the patient responded poorly to plasma expanders and inotropes . while urination began to increase since day 4 , hypotension persisted and was even aggravated by diuresis . since day 25 , his blood pressure became relatively stable , and inotropes were tapered off for 2 weeks . the peak serum creatinine level was 2.2 mg / dl ( 194 mol / l ) on day 6 . laboratory tests at the age of 14 days showed that the plasma renin activity was 22.3 ng / ml / h [ 6 ng / l / s , normal , < 15 ng / ml / h ( < 4 ng / l / s ) ] , serum ace < 5 u / l ( normal , 8.321.4 u / l ) , plasma angiotensin i 2114 pg / ml [ 2114 ng / l , normal , < 180 pg / ml ( < 180 ng / l ) ] , plasma angiotensin ii 61 pg / ml [ 61 ng / l , normal , < 50 pg / ml ( < 50 ng / l ) ] and serum aldosterone 371 pg / ml [ 371 ng / l , normal , 5194 pg / ml ( 5194 ng / l ) ] . mutational analysis of the ace gene revealed novel compound heterozygous mutations , c.g776a [ p.arg(cgc)259his(gag ) ] inherited from the mother and c.1454delc [ p.pro(cct)485leu(ctt)fs ] inherited from the mother . at the age of 1 month , oral fludrocortisone treatment ( 0.1 mg / day ) was started to correct intermittent hyponatremia and hypokalemia . the baby was discharged at the age of 4 months with a serum creatinine level of 0.6 mg / dl ( 53 umol / l ) . the patient is currently 2 years old with normal blood pressure and serum electrolyte levels and mild impairment of renal function [ serum creatinine 0.5 mg / dl ( 44 mol / l ) and estimated glomerular filtration rate 69 ml / min/1.73 m ( 1.15 ml / s/1.73 m ) ] . his weight and height are below the third percentile for his age , but his motor and cognitive functions are normal . to date , five cases surviving the neonatal period of ar - rtd have been reported [ 58 ] . all of the patients except patient 3 had one or more affected siblings , all of which died during the perinatal period . although all of the patients subsequently developed chronic kidney disease , their psychomotor and cognitive development was normal . case reports of ar - rtd patients surviving the neonatal perioda ckd , chronic kidney disease ; tpl , kidney transplantation ; pd , peritoneal dialysis ; tx , treatment . the patient 's elder sibling had been born at 33 weeks gestation and died of respiratory impairment just 15 h after birth . however , no autopsy was performed . spontaneous ileal perforation could have resulted from low perfusion pressure , and another case of rtd with multiple ileal perforation has previously been described . hypocalvaria is also the consequence of low blood pressure because skull membranous bones require high oxygen tension for normal growth [ 4 , 10 ] . renal hypoperfusion is probably the cardinal lesion leading to ar - rtd because the same tubular lesions can be produced secondarily by various fetal conditions associated with insufficient renal blood supply and consequent marked stimulation of the ras , including renal artery stenosis and fetal exposure to ras blockers . therefore , the presumed consequence of all mutations observed in ar - rtd is the absence of angiotensin ii production or function . however , the profiles of ras components vary according to the underlying genetic defect of individual patient . a patient with ace mutations revealed a high plasma renin activity , high active renin concentration and low ace concentration , as shown in the present case . in addition , the present case revealed markedly increased angiotensin i level with mildly increased angiotensin ii and aldosterone levels . the interpretation of the hormonal changes in the present case may be as follows : ( i ) production of angiotensin i , the substrate of ace , is markedly increased to overcome the defective ace activity , ( ii ) the missense ( p.r259h ) mutant ace has minimal residual function or other proteolytic enzyme systems are activated due to defective ace function and ( iii ) a small portion of markedly increased angiotensin i is converted to angiotensin ii by minimally functioning mutant ace or via other proteolytic enzyme systems ( figure 1 ) . the compensatory increase of angiotensin ii may be the cause of survival and milder course of the patient . schreiber et al . recommended an early trial of mineralocorticoids to overcome extreme hypotension and hyperkalemia in patients with rtd . possible sequential changes in the renin angiotensin aldosterone system in the present case . decreased or absent ace activity due to genetic mutations , compensatory overproduction of angiotensin i , the substrate of ace and conversion of a small portion of angiotensin i to angiotensin ii by minimally functioning mutant ( p.r259h ) ace or via other proteolytic enzyme systems such as chymases and tissue plasminogen activators ( t - pa ) . in conclusion , ar - rtd is not a uniformly fatal disease , although all of the surviving cases subsequently developed chronic kidney disease . collection of more surviving cases is required to find out possible prognostic factors and to develop effective treatment .

purposethis study evaluated mid - term results of anterior cruciate ligament ( acl ) reconstruction using the pinn - acl crosspin system that allowed for short graft fixation.materials and methodsforty - three patients underwent single - bundle acl reconstruction with a 4-strand semitendinosus tendon graft using the pinn - acl crosspin system . femoral fixation was done using the pinn - acl crosspin system , and the tibial side was fixed with post - tie and a bioabsorbable interference screw . the mean follow - up period was 50 months . evaluation was done using the lachman test , pivot - shift test , international knee documentation committee ( ikdc ) score and grade . anterior displacement was assessed.resultsthere was improvement in the lachman test and pivot - shift test at final follow - up , form grade ii ( n=40 ) or iii ( n=3 ) to grade i ( n=3 ) or 0 ( n=40 ) and from grade i ( n=20 ) or ii ( n=10 ) to grade i ( n=8 ) or 0 ( n=22 ) , respectively . the mean ikdc score was 88.7 , and grade a and b were 93.0% at final follow - up . side - to - side difference was improved from 6.7 mm to 2.1 mm at final follow - up . complications occurred in 3 patients , a re - ruptured due to trauma at 2 years after surgery and a deep infection and a superficial infection.conclusionsthe mid - term follow - up results of acl reconstruction with the pinn - acl crosspin system were satisfactory . the pinn - acl crosspin can be considered as a useful instrument for short graft fixation . autosomal - recessive renal tubular dysgenesis ( ar - rtd ) is a rare disorder caused by a genetic defect in the renin angiotensin system . although ar - rtd has typically been known as a lethal disease due to refractory hypotension and renal failure immediately after birth , few cases have reported survival of the neonatal period . we report here an additional case of ar - rtd , who had novel ace mutations and survived over 2 years and provide a review of the five previously reported surviving cases . in conclusion , ar - rtd is not a uniformly fatal disease , although factors affecting the survival remain unknown .

patients with ibd should be periodically examined regarding their dermatologic conditions . in a previous report from iran , the prevalence of dermatological manifestations was reported to be 5.9% in patients with ibd with a higher rate in crohn s disease ( 7.29% ) compared with patients with ulcerative colitis ( 4.07% ) . they were more common in women ( 52% ) than in men ( 48% ) . one of the rare dermatological manifestations in patients with ibd is pyoderma gangrenosum . which is more common in patients with ulcerative colitis . since pyoderma gangrenosum is a rare occurrence , its explicit prevalence is unknown , but generally it has been estimated to occur in 3 - 10 million patients annually . in iran , the prevalence of pyoderma gangrenosum in patients with ulcerative colitis has been reported to be 1.4% . the diagnosis of pyoderma gangrenosum is based on physical examination , and examining the lesions regarding its type , number , size , and location as well as associated symptoms of ulcerative colitis . most pustular lesions in patients with ibd should be considered as pyoderma gangrenosum variants and be treated accordingly . even though histopathologic examination is not deemed diagnostic for pyoderma gangrenosum , skin biopsy should be performed to rule out other conditions simulating pyoderma gangrenosum . pyoderma vegetans is a sign of ibd , but rarely occurs in iranian patients . in case of facing pyoderma vegetans in a patient without significant medical history , the mean age for the onset of dermatological manifestations in iranian patients with ibd is 31 years . the patient is a known case of ulcerative colitis limited to her left colon for 15 years under irregular treatments . she was receiving unknown herbal medications for a long time and discontinued her standard treatments in the past three years . she had intermittently developed skin lesions diagnosed as pyoderma gangrenosum in her shoulder , thigh , and genital areas during the past 4 years ( figure 1 ) . ( a ) pyoderma gangrenosum on the right thigh , ( b ) pyoderma gangrenosum on the shoulder , ( c ) pyoderma gangrenosum on the genital area six months before admission to our center , she developed a small mucosal lesion in her nose . she was referred to shariati hospital gastrointestinal clinic because of the rapid growth of the lesions and unresponsiveness to local treatments used by her dermatologist . the only remarkable findings were old skin scars on her extremities and genital area and a lobular dermato - mucosal lesion measured 345 cm in her left nasal lumen ( figure 2 ) . ( a ) the dermato - mucosal lesion in her nose in the first month , ( b ) the dermato - mucosal nasal lesion in month 6 , ( c ) scar of the skin lesion after surgery laboratory assays showed white blood count ( wbc)=6,300/mm , hemoglobin=10.6gr / dlit , platelet count=405,000/mm , esr=61 mm 1st hr , crp=37mg / l with normal stool smear and culture . during the preceding 6 months , the dermatologist who referred the patient had injected corticosteroids into the lesion several times . oral cyclosporine followed by dapsone was administered by the dermatologist with no significant effect on her nasal lesion . since the lesion was large and patient complained of respiratory difficulty , surgical intervention was done and the lesion was excised completely . ( a , b ) microscopic features of the verrucous plaques are acanthotis and papillomatous hyperplasia of epidermis along with multiple micro - abscesses ( pustules ) in dermis ( h&e stain , original magnification 60x ) , ( c , d ) higher power view of the lesion shows acanthotic papillomatous epidermis and subepidermal micro - abscess ( pustule ) ( h&e stain , original magnification 150x ) . no evidence was detected in favor of cytomegalovirus or clostridium difficile on laboratory or pathological examination . after initial evaluation and ruling out latent infections such as tuberculosis , intravenous infliximab ( 300 mg ) was started on weeks 0 , 2 , and 6 and then every 8 weeks . simultaneously oral asacol ( 4.8 grams per day ) was started and she underwent regular monitoring every 8 weeks until week 48 of injection . after 2 months , the clinical symptoms improved significantly and in a 1-year follow - up no recurrence of the nasal lesion was found . in case of simultaneous pyoderma gangrenosum and ulcerative colitis , treatments are delivered directly targeting the skin lesions as well as systemic agents to control ulcerative colitis . topical therapeutics include highly potent steroids , calcineurin inhibitors ( tacrolimus , pimecrolimus ) , human platelet releasing growth factor , and intralesional injection of cyclosporine . systemic treatment for pyoderma gangrenosum involves cyclosporine ( 3 - 5 mg / kg / day ) which is effective for extensive pyoderma gangrenosum and is usually administered with steroids . in most cases , cyclosporine could be applied instead of steroids . dapsone ( maximum dose of 200 mg / day ) is effective in mild cases . when the patient does not respond to routine treatments , infliximab which is a chimeric anti - tumor necrosis factor alpha antibody is an appropriate option . however , it is mandatory that the patient be evaluated regarding latent / active infections such as tuberculosis and be monitored during treatment with infliximab in terms of appearance of infective signs . despite various treatments reported in the literature , an exact treatment guideline does not exist for pyoderma gangrenosum . in the presented patient , in view of the presence of active colitis ( evident in colonoscopy ) and mucosal lesion of pyoderma vegetans in her nose associated with other dermatologic manifestations of pyoderma gangrenosum , intravenous infliximab ( 300 mg , 5 mg / kg ) with oral asacol ( 4.8 gr per day ) was initiated . after 2 months , complete resolution of symptoms as well as improvement in macroscopic and microscopic views in colonoscopy was observed . in a one - year follow - up , it seems that infliximab was effective not only for her ulcerative colitis signs , but also on extra - intestinal manifestation ( i.e. , pyoderma vegetans ) . sepsis is the main cause of mortality in pediatric intensive care units ( picus ) . it is caused by numerous infectious agents , inducing multiple organ dysfunction syndrome ( mods ) , multiple organ failure ( mof ) , and even death . several studies have demonstrated that the severity of sepsis , as well as early diagnosis and prognosis were directly related to mortality . early diagnosis and prognosis are essential to effectively control sepsis , prevent the incidence of mods or mof , and reduce mortality in children with sepsis . currently , the second - generation pediatric index of mortality ( pim-2 ) or pediatric risk of mortality score ( prism ) are used to assess the severity and prognosis of children with sepsis internationally , while pediatric critical illness score ( pcis ) is more commonly used in china . pcis is based on patients heart rate , blood pressure , pao2 , ph , na , k , cr , and hb . a lower pcis score indicates higher disease severity . in addition , a few non - specific inflammatory markers , such as cd15s , nt - probnp , soluble urokinase plasminogen activator receptor ( supar ) , procalcitonin ( pct ) , high - sensitivity c - reactive protein ( hs - crp ) , and pancreatic stone protein ( psp ) are established markers for prognostic evaluation of patients with sepsis [ 1012 ] . baseline procalcitonin levels are linked to severity of pediatric sepsis , while the persistent elevation in procalcitonin despite therapy are associated with increased mortality risk scores . crp , which is one of the most widely available , most studied , and most used laboratory tests for bacterial infection , has the best diagnostic accuracy when combined with another infection marker during the early phases of sepsis . recent studies suggest that psp is a possible biomarker of multiorgan failure and mortality in sepsis . however , its prognostic value in children with sepsis is not entirely clear . in this study , we conducted a prospective analysis of 214 children with sepsis , to investigate the prognostic value of pct , hs - crp , and psp . we enrolled 214 children with sepsis admitted to intensive care units ( icu ) of our hospital between march 2014 and october 2015 . every patient was diagnosed with sepsis according to clinical criteria defined in international sepsis definitions conference in 2001 . severe sepsis was diagnosed when organ dysfunction , hypoperfusion , or hypotension including lactic acidosis , oliguria , or acute altered mental status occurred in septic patients . patients enrolled included 99 males and 115 females , with an average age of 4.61.5 years . all the patients in this study signed informed consent , which was approved by the ethics committee of the first people s hospital of yichang . age , sex , body height , weight , body mass index ( bmi ) , blood pressure , and surgical history were recorded by specialists in the icu . pcis was evaluated by two specialists . if the two scores differed by more than 5 points , another icu physician was invited to perform the final assessment . supernatants were obtained after centrifugation ( 4c , 3,000 rev / minutes , 10 minutes ) and stored at 80c until further analysis . the serum levels of pct and hs - crp were tested by microparticle enzyme immunoassay ( meia ) . continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were displayed as counts or percentages . student s t - test was used for the analysis of continuous variables and -test for categorical variables ; p<0.05 was considered significant . spearman correlation was used to analyze the relationship between pct , hs - crp , psp , and pcis . multivariate logistic regression was used to analyze the risk factors for 28-day mortality in patients with sepsis . receiver operating characteristic ( roc ) analysis was used to compare the prognostic value of pct , hs - crp , and psp in children with sepsis . furthermore , standard indices of validity , such as youden index , sensitivity , and specificity were calculated based on the roc results . we enrolled 214 children with sepsis admitted to intensive care units ( icu ) of our hospital between march 2014 and october 2015 . every patient was diagnosed with sepsis according to clinical criteria defined in international sepsis definitions conference in 2001 . severe sepsis was diagnosed when organ dysfunction , hypoperfusion , or hypotension including lactic acidosis , oliguria , or acute altered mental status occurred in septic patients . patients enrolled included 99 males and 115 females , with an average age of 4.61.5 years . all the patients in this study signed informed consent , which was approved by the ethics committee of the first people s hospital of yichang . age , sex , body height , weight , body mass index ( bmi ) , blood pressure , and surgical history were recorded by specialists in the icu . pcis was evaluated by two specialists . if the two scores differed by more than 5 points , another icu physician was invited to perform the final assessment . supernatants were obtained after centrifugation ( 4c , 3,000 rev / minutes , 10 minutes ) and stored at 80c until further analysis . the serum levels of pct and hs - crp were tested by microparticle enzyme immunoassay ( meia ) . continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were displayed as counts or percentages . student s t - test was used for the analysis of continuous variables and -test for categorical variables ; p<0.05 was considered significant . spearman correlation was used to analyze the relationship between pct , hs - crp , psp , and pcis . multivariate logistic regression was used to analyze the risk factors for 28-day mortality in patients with sepsis . receiver operating characteristic ( roc ) analysis was used to compare the prognostic value of pct , hs - crp , and psp in children with sepsis . furthermore , standard indices of validity , such as youden index , sensitivity , and specificity were calculated based on the roc results . a total of 214 patients were enrolled in this study , with an average age of 4.61.5 years , and including 99 males and 115 females . after a follow - up of 28 days , no significant differences in patients age , sex , or weight were found between the dying and surviving groups of patients . pcis scores in the dying patients were lower than in the surviving group ( p<0.001 ) . further , the serum pct , hs - crp , and psp levels were higher in the dying group than in the surviving group of patients ( p<0.001 ; table 1 ) . as shown in figure 1 , pct was negatively correlated with pcis , r=0.4474 ( p<0.001 ; figure 1a ) ; hs - crp was negatively correlated with pcis , significantly ( r=0.3479 , p<0.001 ; figure 1b ) ; and psp showed a distinctly negative correlation with pcis ( figure 1c ) . the results indicated that the levels of pct , hs - crp , and psp were correlated with disease severity . as shown in table 2 , multivariate logistic regression analysis revealed pcis as a protective factor in the 28-day mortality of children with sepsis ( or=0.79 ; 95% ci=0.670.89 ) . furthermore , psp ( or=2.38 , 95% ci=1.465.76 ) was more sensitive than pct ( or=1.34 , 95% ci=1.022.25 ) , p=0.0031 . the serum concentrations of pct , hs - crp , and psp were higher in the dying group of patients ( p<0.01 ; figures 24 ) . to further determine the prognostic value of the three markers in children with sepsis , receiver operating characteristic ( roc ) curves were used to evaluate the predictive power . the results indicated that area under the curve ( auc ) values of pct , hs - crp , and psp were 0.83 ( 95% ci , 0.770.88 ) , 0.76 ( 95% ci , 0.700.82 ) and 0.73 ( 95% ci , 0.670.79 ) , respectively . as illustrated in table 3 , the sensitivity and specificity of cutoff values were calculated according to roc curve analysis . subsequently , the roc curve comparison revealed a higher prognostic value of pct compared with hs - crp and psp ( p<0.001 ) as shown in figure 5 . multivariate logistic regression was conducted to calculate the coefficients of these biomarkers when used in predicting mortality in patients with sepsis ( pct & hs - crp & psp ) = 12.3125 + 0.068404*pct + 0.058065*hs - crp + 0.012057*psp . the results of roc analysis of ( pct & hs - crp & psp ) are shown in table 3 . the prognostic value of the combined pct , hs - crp , and psp levels in children with sepsis was higher than the individual levels ( p<0.001 ; figure 5 ) . a total of 214 patients were enrolled in this study , with an average age of 4.61.5 years , and including 99 males and 115 females . after a follow - up of 28 days , no significant differences in patients age , sex , or weight were found between the dying and surviving groups of patients . pcis scores in the dying patients were lower than in the surviving group ( p<0.001 ) . further , the serum pct , hs - crp , and psp levels were higher in the dying group than in the surviving group of patients ( p<0.001 ; table 1 ) . as shown in figure 1 , pct was negatively correlated with pcis , r=0.4474 ( p<0.001 ; figure 1a ) ; hs - crp was negatively correlated with pcis , significantly ( r=0.3479 , p<0.001 ; figure 1b ) ; and psp showed a distinctly negative correlation with pcis ( figure 1c ) . the results indicated that the levels of pct , hs - crp , and psp were correlated with disease severity . as shown in table 2 , multivariate logistic regression analysis revealed pcis as a protective factor in the 28-day mortality of children with sepsis ( or=0.79 ; 95% ci=0.670.89 ) . furthermore , psp ( or=2.38 , 95% ci=1.465.76 ) was more sensitive than pct ( or=1.34 , 95% ci=1.022.25 ) , p=0.0031 . the serum concentrations of pct , hs - crp , and psp were higher in the dying group of patients ( p<0.01 ; figures 24 ) . to further determine the prognostic value of the three markers in children with sepsis , receiver operating characteristic ( roc ) curves were used to evaluate the predictive power . the results indicated that area under the curve ( auc ) values of pct , hs - crp , and psp were 0.83 ( 95% ci , 0.770.88 ) , 0.76 ( 95% ci , 0.700.82 ) and 0.73 ( 95% ci , 0.670.79 ) , respectively . as illustrated in table 3 , the sensitivity and specificity of cutoff values were calculated according to roc curve analysis . subsequently , the roc curve comparison revealed a higher prognostic value of pct compared with hs - crp and psp ( p<0.001 ) as shown in figure 5 . multivariate logistic regression was conducted to calculate the coefficients of these biomarkers when used in predicting mortality in patients with sepsis ( pct & hs - crp & psp ) = 12.3125 + 0.068404*pct + 0.058065*hs - crp + 0.012057*psp . the results of roc analysis of ( pct & hs - crp & psp ) are shown in table 3 . the prognostic value of the combined pct , hs - crp , and psp levels in children with sepsis was higher than the individual levels ( p<0.001 ; figure 5 ) . it develops into severe sepsis or septic shock , resulting in mods , mof , or even death . interestingly , recent studies suggest that nonspecific inflammation and abnormal expression of inflammatory cytokines rather than microbial infection leads to organ damage . early diagnosis and intervention has been shown to reduce the risk of sepsis - related mortality . in our study , the overall mortality was relatively high , which might be due to the high percentage ( 40.5% ) of post - surgery patients . its diagnostic and predictive value in patients with sepsis has been confirmed in several studies . found that serum pct was an early systemic marker of sepsis , which was correlated closely with mortality and inversely with serum calcium in bacterial peritonitis of hamster . found that serum pct increased sharply during early sepsis , and that the persistent levels decreased only after effective antibiotic treatment . in addition , nakamura et al . found that pct level was a good indicator of the severity of infection , with prognostic value in 393 adult patients with sepsis . similarly , zurek et al . reported that serum levels of pct were positively correlated with prognosis of pediatric sepsis . our results showed that pct levels were significantly higher in the dying patients than in the surviving group , and were negatively correlated with pcis , reminding us that pct was related to the severity of sepsis . furthermore , the results of roc curve analysis indicated that an auc value of 0.83 ( 95% ci , 0.770.88 , supported the prognostic value of pct in children with sepsis . high - sensitivity c - reactive protein ( hs - crp ) is a key inflammatory cytokine , which is present in small amounts under normal conditions . plasma hs - crp is an early marker of sepsis severity and poor prognosis , and is one of the risk factors for cardiovascular disease . pancreatic stone protein ( psp ) belongs to the family of lectin - binding proteins , and is constitutively secreted by pancreatic acinar cells into pancreatic juice along with zymogens . palmiere et al . reported that psp and pct were positively correlated with mortality in patients with or without sepsis , and that the psp levels were distinctly higher than in sepsis . peng et al . observed a significant positive correlation between psp and wbc , as well as serum pct levels . dynamic monitoring of psp was used to evaluate a patient s condition and assess their risk of death . our study showed that serum hs - crp and psp levels increased in dying patients and were negatively correlated with pcis . these results indicated that serum hs - crp and psp levels were correlated with the severity of sepsis . furthermore , multivariate logistic regression analysis revealed that both hs - crp and psp were independent risk factors for pediatric sepsis , with hrs of 1.79 ( 95% ci , 1.312.42 ) and 2.38 ( 95% ci , 1.465.76 ) . in addition , roc analysis showed that both hs - crp and psp levels were appropriate for clinical application . it is unlikely that any single biomarker is a predictor of outcomes in the pediatric population . the complexity of interactions driving host immune response and genetic variation suggests that multiple biomarkers are involved in predicting outcomes . stratification of pediatric patients based on their genome expression is a reasonable approach to identify high- and low - risk groups . in our study , we used multivariate logistic regression to calculate each index before using the three markers : ( pct & hs - crp & psp ) = 12.3125 + 0.068404*pct + 0.058065*hs - crp + 0.012057*psp . the roc curve analysis yielded an auc value ( pct & hs - crp & psp ) up to 0.92 , which was clearly superior to pct , hs - crp , or psp alone . technological advances facilitate testing of multiple inflammatory cytokines rapidly and sensitively using small amounts of serum sample . therefore , the joint detection of pct , hs - crp , and psp clearly improves the sensitivity and specificity of prognosis in children with sepsis , and represents a useful serum biomarker . our study found that serum pct , hs - crp , and psp levels were promising biomarkers of risk and useful clinical tools for risk stratification of pediatric sepsis . furthermore , the combination of serum pct , hs - crp , and psp is potentially a very useful biomarker with high sensitivity and specificity , and remarkable prognostic value in pediatric sepsis .

some dermatologic manifestations are common in ulcerative colitis ( uc ) . herein , we present a 36-year - old woman with ulcerative colitis and uncommon nasal mucosa pyoderma vegetans . the patient presented to our hospital with symptoms of active colitis and a concomitant 345 cm dermato - mucosal lesion in her left nasal lumen . after surgery of the mucosal lesion , the treatment for her active colitis was initiated with intravenous infliximab and oral asacol . after a 1-year follow - up , no sign of recurrence favoring mucosal lesion was noted and symptoms of ulcerative colitis were managed properly . backgroundto investigate the prognostic value of procalcitonin ( pct ) , high - sensitivity c - reactive protein ( hs - crp ) , and pancreatic stone protein ( psp ) in children with sepsis.material/methodsa total of 214 patients with sepsis during hospitalization were enrolled . serum levels of pct , hs - crp , and psp were measured on day 1 of hospitalization and the survival rates of children were recorded after a follow - up of 28 days . pearson s correlation analysis was conducted to test the association of pct , hs - crp , and psp with pediatric critical illness score ( pcis ) . logistic regression models were used to analyze the risk factors contributing to patients death . the auc was used to determine the value of pct , hs - crp , and psp in the prognosis of patients with sepsis.resultsthe expression of pct , hs - crp , and psp in the dying patients was higher than in the surviving patients ( p<0.001 ) . pearson s correlation analysis showed that serum pct , hs - crp , and psp levels were negatively correlated with pcis ( p<0.001 ) . multivariate logistic regression revealed that pct , hs - crp , and psp were independent risk factors for the prognosis of patients with sepsis ( p<0.001 ) . roc analysis showed the auc values of pct , hs - crp , and psp were 0.83 ( 95% ci , 0.770.88 ) , 0.76 ( 95% ci , 0.700.82 ) , and 0.73 ( 95% ci , 0.670.79 ) , respectively . the combined auc value of pct , hs - crp , and psp , was 0.92 ( 95% ci , 0.870.95 ) , which was significantly increased compared with pct , hs - crp , or psp ( p<0.001).conclusionsthe combination of serum pct , hs - crp , and psp represents a promising biomarker of risk , and is a useful clinical tool for risk stratification of children with sepsis .

rna can play an important functional role in catalysis , e.g. ribozymes are rna enzymes that cleave rna phosphodiester bonds at specific sites ( 1 ) ; see ( 2 ) for an overview of potential therapeutic applications of ribozymes to cleave mrnas of oncogenes ( ras or bcr - abl ) and viral transcripts ( hiv-1 ) , to overcome drug resistance , control arthritis , etc . such is the case for the aminoglycoside and macrolide families of antibiotics , which disrupt rna translation in prokaryotes by targeting ribosomal ( rrna ) ( 3 ) . in contrast to mrna , noncoding rna ( ncrna ) is transcribed from genomic dna and plays a biologically important role , although it is not translated into protein . examples of ncrna include ribozymes , riboswitches , micro rna , small interfering rna ( 4 ) , trna , rrna , etc . riboswitches have recently been discovered to interact with small ligands and up- or down - regulate certain genes . breaker and co - workers ( 5 ) report the crystal structures of the add a - riboswitch and xpt g - riboswitch aptamer modules , which distinguish between bound adenine and guanine ; see ( 6 ) for an overview of bacterial riboswitches , and ( 7 ) for the structure , as given in the pdb code 1u8d of a guanine - responsive riboswitch with the metabolite hypoxanthine . rnaomics ( 8) , analogous to proteomics , concerns aspects of the secondary and tertiary structure , folding pathway , kinetics , comparison , function and regulation of all rna in a living organism . rnaomics requires the application of numerous existent tools , as well as the development of new computational methods . well - known rna computational tools include secondary structure prediction web servers mfold ( 9 ) and vienna rna package ( 10 ) , the sfold web server ( 11 ) to sample secondary structures according to the boltzmann probability distribution , the trnascan - se gene finder for trna ( 12 ) , multiple sequence alignment for the statistical detection of rna secondary structure msari ( 13 ) , dynamic programming pairwise sequence - structure alignment dynalign ( 14 ) , tertiary structure modeling tool mc - sym ( 15 ) , etc . only a few of the many important computational tools for rna structure prediction , gene finding , alignment , etc . have been listed . in this paper , we describe the web server rnaloss , based on the algorithm of clote ( 16 ) , which computes an aspect of the folding landscape of an rna nucleotide sequence s = s1 , , sn . given s , this algorithm runs in time o(n ) and space o(n ) , and computes for each k , the number of k - locally optimal secondary structures ( explained below ) . work by clote ( 16 ) was motivated by the following question , as has been suggested for proteins ( 17 ) : is it the case that rna has been under selective pressure to fold rapidly ? using the algorithm of the web server rnaloss , it appears that structural rna has a different folding landscape than random rna of the same dinucleotide frequency ; specifically , for small values of k , there appear to be fewer k - locally optimal secondary structures than in random rna . related , but distinct work has appeared in ( 1821 ) , for discussion see ( 16 ) . a secondary structure for an rna sequence s = s1, ,sn is an expression s = s1, ,sn involving dot , left and right parenthesis , which is well balanced , such that nucleotides corresponding to matching parentheses are either watson crick complements or gu wobble pairs . a secondary structure s on rna sequence s = s1, ,sn is defined to be a set of ordered pairs ( i , j ) , such that i + 3 < j and the following conditions are satisfied . watson crick or gu wobble pairs : if ( i , j ) belongs to s , then pair ( ai , aj ) must be one of the following canonical base pairs : ( a , u ) , ( u , a ) , ( g , c ) , ( c , g ) , ( g , u ) and ( u , g).threshold requirement : if ( i , j ) belongs to s , then j i > 3 ; i.e. there must be at least three unpaired bases in a hairpin loop.non-existence of pseudoknots : if ( i , j ) and ( k , l ) belong to s , then it is not the case that i < k < j < l.no base triples : if ( i , j ) and ( i , k ) belong to s , then j = k ; if ( i , j ) and ( k , j ) belong to s , then i = k. watson crick or gu wobble pairs : if ( i , j ) belongs to s , then pair ( ai , aj ) must be one of the following canonical base pairs : ( a , u ) , ( u , a ) , ( g , c ) , ( c , g ) , ( g , u ) and ( u , g ) . threshold requirement : if ( i , j ) belongs to s , then j i > 3 ; i.e. there must be at least three unpaired bases in a hairpin loop . non - existence of pseudoknots : if ( i , j ) and ( k , l ) belong to s , then it is not the case that i < k < j < l. no base triples : if ( i , j ) and ( i , k ) belong to s , then j = k ; if ( i , j ) and ( k , j ) belong to s , then i = k. a secondary structure is k - locally optimal if it has k fewer base pairs than the maximum possible number [ i.e. than in the nussinov jacobson optimal structure ( 22,23 ) ] , and yet no base pairs can be added without violating the definition of secondary structure ( e.g. without introducing a pseudoknot ) . to illustrate this notion , consider the rna sequence ggggccccc , which has three as the maximum possible number of base pairs , as given in the structure ( ( ( ) ) ) . there is only one structure having 3 bp , so the number of 0-locally optimal secondary structures is 1 . on the other hand , the latter are listed as follows : ( i ) ( ) .... ( ii ) ( .... ) .. ( iii ) ( ..... ) . the algorithm of ( 16 ) uses dynamic programming to compute , for each i < j and each k , the number of k - locally optimal secondary structures on the subsequence s = si, ,sj . additionally , the algorithm must keep track of visible nucleotides and positions , i.e. those external to any base pair [ for technical details see ( 16 ) ] . a secondary structure s on rna sequence s = s1, ,sn is defined to be a set of ordered pairs ( i , j ) , such that i + 3 < j and the following conditions are satisfied . watson crick or gu wobble pairs : if ( i , j ) belongs to s , then pair ( ai , aj ) must be one of the following canonical base pairs : ( a , u ) , ( u , a ) , ( g , c ) , ( c , g ) , ( g , u ) and ( u , g).threshold requirement : if ( i , j ) belongs to s , then j i > 3 ; i.e. there must be at least three unpaired bases in a hairpin loop.non-existence of pseudoknots : if ( i , j ) and ( k , l ) belong to s , then it is not the case that i < k < j < l.no base triples : if ( i , j ) and ( i , k ) belong to s , then j = k ; if ( i , j ) and ( k , j ) belong to s , then i = k. watson crick or gu wobble pairs : if ( i , j ) belongs to s , then pair ( ai , aj ) must be one of the following canonical base pairs : ( a , u ) , ( u , a ) , ( g , c ) , ( c , g ) , ( g , u ) and ( u , g ) . threshold requirement : if ( i , j ) belongs to s , then j i > 3 ; i.e. there must be at least three unpaired bases in a hairpin loop . non - existence of pseudoknots : if ( i , j ) and ( k , l ) belong to s , then it is not the case that i < k < j < l. no base triples : if ( i , j ) and ( i , k ) belong to s , then j = k ; if ( i , j ) and ( k , j ) belong to s , then i = k. a secondary structure is k - locally optimal if it has k fewer base pairs than the maximum possible number [ i.e. than in the nussinov jacobson optimal structure ( 22,23 ) ] , and yet no base pairs can be added without violating the definition of secondary structure ( e.g. without introducing a pseudoknot ) . to illustrate this notion , consider the rna sequence ggggccccc , which has three as the maximum possible number of base pairs , as given in the structure ( ( ( ) ) ) . there is only one structure having 3 bp , so the number of 0-locally optimal secondary structures is 1 . on the other hand , the latter are listed as follows : ( i ) ( ) .... ( ii ) ( .... ) .. ( iii ) ( ..... ) . the algorithm of ( 16 ) uses dynamic programming to compute , for each i < j and each k , the number of k - locally optimal secondary structures on the subsequence s = si, ,sj . additionally , the algorithm must keep track of visible nucleotides and positions , i.e. those external to any base pair [ for technical details see ( 16 ) ] . the web server rnaloss implements a new algorithm , described in ( 16 ) , running in o(n ) time and o(n ) space , which computes for a given rna sequence s = s1, ,sn and all k 0 , the number of k - locally optimal secondary structures for s. an rna nucleotide sequence may be input by uploading a fasta - format file or by entering a nucleotide sequence in the blank provided on the web server form . three tables are returned by rnaloss : the number of k - locally optimal secondary structures , the relative density of states ( i.e. the ratio of number of k - locally optimal structures over the total number of locally optimal structures ) and the minimum free energy ( mfe ) of a sample k - locally optimal secondary structure ( for each value of k , rnaloss computes a single k - locally optimal secondary structure , denoted here as sk , among the many possible k - locally optimal structures . since this feature was implemented for debugging purposes , the current version of rnaloss does not guarantee that sk has lowest mfe as evaluated by rnaeval , over all k - locally optimal secondary structures . for this reason , the energy of sample structures sk does not necessarily increase monotonically with increasing value of k ) . for the latter , mfe is computed using rnafold from the vienna rna package . figure 2 lists the number of k - locally optimal secondary structures as computed by rnaloss for type iii hammerhead ribozyme af170517 from rfam ( 24 ) . figure 3 presents the relative density of states for k - locally optimal secondary structures for af170517 . owing to algorithmic time and space constraints , the rnaloss web server immediately processes rna of length at most 60 nt , while for rna of length 61100 nt , the results are emailed to the user . currently , rnaloss refuses to process any sequence of length > 100 nt . current hardware supporting rnaloss web server consists of a beowulf - style cluster comprising 6 dell 1650 , 2 1300 mhz pentium iii , 2 gb ram with 4 apple xserve , 2 1333 mhz g4 , 2 gb ram and finally 6 dell 1850 , 2 2800 mhz xeon em64 t , 2 gb ram . pentium iii nodes are running redhat linux 9 , xeon em64 t nodes are running whitebox linux 3 and g4 nodes are running macos 10.2.8 . upon testing , structurally important rna , such as selenocysteine insertion sequence elements , precursor mrnas , type iii hammerhead ribozymes and trna , all have a markedly smaller number of k - locally optimal structures than that of random rna of the same dinucleotide frequency , for small and moderate values of k. since the free energy of k - locally optimal secondary structures is generally closer to that of the native state for small k , this suggests that structural rna has been optimized not only to have low folding energy ( 25 ) , but also to have relatively few potential kinetic traps . this suggests that rnaloss might be of use in designing rna sequences for rapid folding . users may input an rna sequence consisting of upper or lower case nucleotides a , c , g , t , u , either by uploading a fasta - format file ( a ) or by pasting a nucleotide sequence into ( b ) . output of web server rnaloss on type iii hammerhead ribozyme af170517 from rfam ( 24 ) . the web server rnaloss outputs a table of number of k - locally optimal secondary structures , for each possible value of k 0 ( shown here ) . additionally , tables for the relative density of states and mfe values of sample k - locally optimal secondary structures are displayed in the browser ( data not shown ) . column graph of relative density of states is obtained by clicking a hot link from the previous screen shot . for each value of k 0 , the ratio of number of k - locally optimal secondary structures over the total number of locally optimal secondary structures is displayed . central neuropathic pain ( central pain ) is pain caused by a disease or lesion in the central nervous system . central pain develops in about 8% of stroke patients ( andersen et al 1995 ) , 25% of patients with multiple sclerosis ( osterberg et al 2005 ) , and 40%50% of patients with spinal cord injury ( budh et al 2003 ; siddall et al 2003 ; werhagen et al 2004 ) and may develop secondary to brain and spinal cord tumors and other diseases affecting the central nervous system . central pain thus affects a large number of patients worldwide and often it has a substantial impact on the quality of life , mood , sleep , cognition , social relations , etc . central pain is characterized by ongoing pain , which may be burning , squeezing , pricking , and shooting and/or evoked types of pain , eg , pain evoked by light touch . the pain is located within an area of sensory disturbance covering various proportions of the deafferented body regions . treatment of central pain is often difficult and requires a different approach than nociceptive pain . central pain is usually treated with antidepressants , anticonvulsants , and opioids ; treatments which provide partial pain relief at best and which are often associated with side - effects . pregabalin is a novel , centrally acting neuromodulating agent that was approved by the us food and drug administration ( fda ) in 2004 for the treatment of painful diabetic peripheral neuropathy and post - herpetic neuralgia . in 2005 it was approved as adjunctive therapy in adults with partial seizures and recently it has been approved for the treatment of fibromyalgia . pregabalin is approved by the european medicines agency ( emea ) for the treatment of peripheral and central neuropathic pain in adults , as adjunctive therapy in adults with partial seizures , and for the treatment of generalized anxiety disorder ( gad ) in adults ( emea 2006 ; pfizer 2007 ) . pregabalin ( ( s)-3-(aminomethyl)-5-methylhexanoic acid ) is a structural derivative of the inhibitory neurotransmitter -aminobutyric acid ( gaba ) . pregabalin is structurally related to gabapentin and has a similar pharmacological profile and anticonvulsant and analgesic activity ( ben - menachem 2004 ) . the predominant mechanism of action is thought to be through its presynaptic binding to the 2 subunit of voltage - gated calcium channels which in turns leads to reduced release of neurotransmitters , eg , glutamate , substance p , and calcitonin gene - related peptide ( fehrenbacher et al 2003 ; sills 2006 ; li et al 2006 ; dooley et al 2007 ; taylor et al 2007 ) . such decrease in neurotransmitter release from synapses in several neuronal tissues in the spinal cord and brain is likely to attenuate neuronal hyperexcitability and abnormal synchronization and may thus explain its anticonvulsant , analgesic , and anxiolytic activity ( taylor et al 2007 ) . pregabalin does not appear to act through the gabaergic neurotransmitter system ( reviewed in , eg , ( sills 2006 ) and ( taylor et al 2007 ) ) and although it has been shown to act on voltage - gated potassium channels ( mcclelland et al 2004 ) , this mechanism of action is not thought to contribute significantly to the pharmacological profile ( sills 2006 ) . the pharmacokinetic and safety properties of pregabalin have been studied in healthy subjects and patients with renal impairment ( randinitis et al 2003 ) . the oral bioavailability is 90% and dose - independent , and pregabalin is rapidly absorbed in the fasting state with a tmax of 1 hour which is reduced by food consumption by 35% . food does not alter the area under the curve and has no clinically significant effect . pregabalin does not bind to plasma proteins and thus readily penetrates the blood - brain barrier . over 98% of pregabalin the elimination half - time is 4.86.3 hours but is increased in patients with renal impairment and dependent on the creatinine clearance . therefore , dose reduction is needed in patients with impaired renal function ( ie creatinine clearance < 60 ml / min ) ( randinitis et al 2003 ) ( table 1 ) . pregabalin is not metabolized in the liver and has no effect on the cytochrome p450 system or other liver enzymes and has no plasma protein binding consistent with the lack of interactions with other anticonvulsants , certain antidiabetics , and oral contraceptives ( ben - menachem 2004 ; tassone et al 2007 ) . additive adverse effects on cognitive and gross motor functioning have been seen with pregabalin co - administered with oxycodone , lorazepam , and ethanol , and concomitant treatment with pregabalin and a thiazolidinedione anti - diabetic agent may lead to an additive effect on edema and weight gain ( emea 2004 ; pfizer 2007 ) . pregabalin has in large published parallel - group design studies consistently been shown to relieve post - herpetic neuralgia ( dworkin et al 2003 ; sabatowski et al 2004 ; freynhagen et al 2005 ; van seventer et al 2006 ) and painful diabetic neuropathy ( lesser et al 2004 ; rosenstock et al 2004 ; freynhagen et al 2005 ; richter et al 2005 ) with a combined number needed to treat ( nnt ) for doses ranging from 300 mg to 600 mg of 3.9 ( 3.34.7 ) . for comparison , the nnt values in peripheral neuropathic pain are 2.3 ( 2.12.7 ) for tricyclic antidepressants , 2.7 ( 2.13.6 ) for opioids , 3.9 ( 2.76.7 ) for tramadol , 4.4 ( 2.517 ) for topical lidocaine , and 5.5 ( 3.414 ) for serotonin noradrenaline reuptake inhibitors , but differences in design and study population may make direct comparison of nnt values difficult ( finnerup et al 2005 ) . the total number of patients included 1028 exposed to pregabalin and 575 to placebo in the 300600 mg dose range . two studies in painful diabetic neuropathy reported by the european medicines agency in 2004 ( emea 2004 ) are still unpublished ; one study including 396 patients showed efficacy of pregabalin 300/600 mg daily , while a 3-armed study with pregabalin 600 mg ( n = 87 ) , amitriptyline 75 mg ( n = 88 ) and placebo ( n = 81 ) failed to show a significant pain - relieving effect with pregabalin ( p = 0.08 ) . the difference in mean endpoint score between pregabalin and placebo for all peripheral neuropathic pain studies ranged from 0.18 to 1.57 points for the 300 mg daily score and from 0.64 to 2.02 points for the 600 mg daily score ( emea 2004 ) . there was no effect of pregabalin 75 mg daily and inconsistent efficacy for 150 mg daily . the first studies on pregabalin ( dworkin et al 2003 ; lesser et al 2004 ; rosenstock et al 2004 ; sabatowski et al 2004 ) excluded patients who failed to respond to previous treatment with gabapentin , which may bias the efficacy outcome measures in favor of pregabalin ( finnerup et al 2005 ) , but more recent trials without this exclusion criterion have comparable nnt values ( freynhagen et al 2005 ; richter et al 2005 ; van seventer et al 2006 ) . somnolence is a frequent adverse event present in 20%30% and subanalyses have shown that the pain relieving effect was larger in patients experiencing somnolence as an adverse effect ( emea 2004 ) . however , pregabalin still had a pain relieving effect in those patients not experiencing somnolence as an adverse effect ( emea 2004 ) . the published clinical trials found dose - dependent efficacy in pain relief as well as improvements in sleep and global impressions of changes ( integrating the effect of treatment and side - effects ) , and some studies also in quality of life measures ( lesser et al 2004 ; sabatowski et al 2004 ) and mood ( rosenstock et al 2004 ) . two randomized placebo - controlled trials have been conducted in central pain ( table 2 ) . the first study published is a parallel group design study in central neuropathic pain due to spinal cord injury ( siddall et al 2006 ) . a baseline week was followed by a 3-week titration period where pregabalin was increased up to 300 mg bid and a 9-week fixed dose period . concurrent pain medication was kept constant during the trial and included tricyclic antidepressants in 33% in the pregabalin group and 18% in the placebo group , opioids in 30% and 48% , and antiepileptic drugs except gabapentin in 11% and 9 % respectively . muscle relaxants ( including baclofen ) were used by 54% in the pregabalin group and 37% in the placebo group while benzodiazepines were used by 40% and 39% respectively . pain was evaluated daily on a numeric rating scale ( nrs , 010 ) and the primary efficacy measure was the weekly mean pain score at endpoint ( last week on study drug ) . the improvement in pain score from baseline ( pregabalin placebo ) was 1.53 ( 0.92 to 2.15 ) , similar to values observed in studies in peripheral neuropathic pain . the effect was significant from week 1 and remained so for the duration of the study . the nnt for 50% pain relief ( 7.1 ( 3.937 ) ) was higher than in most peripheral neuropathic pain studies ; however , the nnt for 30% pain relief ( 3.9 ( 2.59.1 ) ) and pain improvement on the patient global impression of change ( 2.9 ( 205.1 ) ) was similar to what is observed in post - herpetic neuralgia and painful diabetic neuropathy ( lesser et al 2004 ; rosenstock et al 2004 ; sabatowski et al 2004 ; freynhagen et al 2005 ; van seventer et al 2006 ) . recently , pregabalin was studied in a parallel group design study in patients with central pain following stroke or spinal cord injury ( vranken et al 2007 ) . the etiology was stroke in 19 patients ( of these thalamic lesion in 4 and brainstem infarction in 3 ) and spinal cord injury in 21 patients ( of these 11 had a complete injury ) . the diagnoses were evenly distributed among patients allocated to pregabalin ( n = 20 ) and to placebo ( n = 20 ) . for the diagnosis of central pain , the pain should be described as burning , paroxysmal episodes of shooting pain , or pain on light touch , and patients had to score above 12 on the leeds assessment of neuropathic symptoms and signs questionnaire ( lanss ) ( bennett 2001 ) . a baseline pain score above 6 ( visual analog scale , vas ) was required . in a flexible - dose regime and with no base - line period , patients received escalating doses of either pregabalin tablets 150 mg or matching placebo capsules bid titrated at 3-day intervals until a pain reduction of 1.8 on a vas was obtained , they reached the maximum daily dose of 600 mg , or had intolerable side - effects . the patients then remained on the final dose during the remainder of the study period , which was 4 weeks . patients treated with gabapentin discontinued this treatment at least 3 days before receiving study medication . pain medication that was continued during the trial was opioids in 53% , antidepressants in 30% , and carbamazepine in 10% . seventeen patients in the pregabalin group completed the study : nine received 600 mg and eight received 300 mg daily . the primary efficacy parameter was the pain intensity based on the average of 3 vas pain scores measured during the 24 hours prior to baseline and at the end of the 4-week treatment period . the improvement in pain score from baseline ( pregabalin placebo ) was 2.18 ( 0.57 to 3.80 ) with no difference in efficacy between the groups with spinal and brain injury . the nnt for 50% pain relief was low , 3.3 ( 1.914.3 ) , and for 30% pain relief it was 4.0 ( 2.0328 ) . in both studies , pregabalin was an add - on analgesic , which suggests that responses may be due to synergistic interactions . in painful diabetic neuropathy pregabalin was effective as monotherapy ( lesser et al 2004 ; rosenstock et al 2004 ; richer et al 2005 ) , and in both studies in central pain , effect sizes were similar regardless of whether patients used any concomitant analgesics ( siddall et al 2004 ; vranken et al 2007 ) . this would suggest that pregabalin is effective as monotherapy also in central pain , but potential synergistic effects need to be studied in appropriately designed studies . pregabalin is generally well tolerated with no contraindications except for known hypersensitivity to pregabalin or its components . the most common adverse reactions in the peripheral neuropathic pain studies were dose - related dizziness ( 22%38% ) and somnolence ( 11%25% ) , which does not resolve in about one third of patients . these side - effects pose a risk for accidental injury in the elderly . other adverse reactions were dry mouth , asthenia , blurred vision , ataxia , peripheral edema , and weight gain not limited to patients with edema . pregabalin treatment is not associated with clinical significant withdrawal syndromes ( frampton and foster 2006 ) , but abrupt discontinuation may cause insomnia , nausea , headache , or diarrhea and it is recommended to taper off during at least one week ( pfizer 2007 ) . in case of persistent blurred vision , a visual field testing and funduscopic examination may be considered , and patients are advised to report unexplained muscle pain particularly if accompanied with malaise and fever ( pfizer 2007 ) due to unsettled relation of pregabalin to rhabdomyolysis and creatine kinase elevations . pregabalin is recommended to be used with caution in patients with congestive heart failure ( nyha , ( new york heart association ) class iii and iv ) because of limited data in this population ( pfizer 2007 ) . in the two studies in central pain , the frequency of somnolence in the trial by siddall et al ( 2006 ) ( 41% in the pregabalin group and 9% in the placebo group ) was more common than in studies in peripheral neuropathic pain , which may be attributed to additive effects of concomitant medications such as baclofen and benzodiazepines in this patient population . in the study by vranken et al ( 2007 ) , somnolence occurred in 45% but was equally common in the placebo group . other more frequent adverse reactions in the pregabalin group in the siddall study included : dizziness observed in 24% , edema in 20% , asthenia and dry mouth each in 16% , constipation in 13% , amnesia in 10% , and blurred vision in 9% . the frequency of peripheral edema ( 10% ) was not higher than that observed in peripheral neuropathic pain . two patients in the placebo group and eight in the pregabalin group had a weight gain 7% . the median time to onset of somnolence and dizziness was within 8 and 6 withdrawal due to side - effects occurred in 15 pregabalin- and 9 placebo - treated patients . two adverse reactions were considered related to treatment : one had a withdrawal reaction 1 day following pregabalin discontinuation with increased spasticity and impaired coordination , and one had edema , hypervolemia and reduced platelet count caused by an infection . in the study by vranken et al ( 2007 ) , side - effects were mild to moderate with no difference in frequency of adverse reactions in the two study groups . pregabalin has in large published parallel - group design studies consistently been shown to relieve post - herpetic neuralgia ( dworkin et al 2003 ; sabatowski et al 2004 ; freynhagen et al 2005 ; van seventer et al 2006 ) and painful diabetic neuropathy ( lesser et al 2004 ; rosenstock et al 2004 ; freynhagen et al 2005 ; richter et al 2005 ) with a combined number needed to treat ( nnt ) for doses ranging from 300 mg to 600 mg of 3.9 ( 3.34.7 ) . for comparison , the nnt values in peripheral neuropathic pain are 2.3 ( 2.12.7 ) for tricyclic antidepressants , 2.7 ( 2.13.6 ) for opioids , 3.9 ( 2.76.7 ) for tramadol , 4.4 ( 2.517 ) for topical lidocaine , and 5.5 ( 3.414 ) for serotonin noradrenaline reuptake inhibitors , but differences in design and study population may make direct comparison of nnt values difficult ( finnerup et al 2005 ) . the total number of patients included 1028 exposed to pregabalin and 575 to placebo in the 300600 mg dose range . two studies in painful diabetic neuropathy reported by the european medicines agency in 2004 ( emea 2004 ) are still unpublished ; one study including 396 patients showed efficacy of pregabalin 300/600 mg daily , while a 3-armed study with pregabalin 600 mg ( n = 87 ) , amitriptyline 75 mg ( n = 88 ) and placebo ( n = 81 ) failed to show a significant pain - relieving effect with pregabalin ( p = 0.08 ) . the difference in mean endpoint score between pregabalin and placebo for all peripheral neuropathic pain studies ranged from 0.18 to 1.57 points for the 300 mg daily score and from 0.64 to 2.02 points for the 600 mg daily score ( emea 2004 ) . there was no effect of pregabalin 75 mg daily and inconsistent efficacy for 150 mg daily . the first studies on pregabalin ( dworkin et al 2003 ; lesser et al 2004 ; rosenstock et al 2004 ; sabatowski et al 2004 ) excluded patients who failed to respond to previous treatment with gabapentin , which may bias the efficacy outcome measures in favor of pregabalin ( finnerup et al 2005 ) , but more recent trials without this exclusion criterion have comparable nnt values ( freynhagen et al 2005 ; richter et al 2005 ; van seventer et al 2006 ) . somnolence is a frequent adverse event present in 20%30% and subanalyses have shown that the pain relieving effect was larger in patients experiencing somnolence as an adverse effect ( emea 2004 ) . however , pregabalin still had a pain relieving effect in those patients not experiencing somnolence as an adverse effect ( emea 2004 ) . the published clinical trials found dose - dependent efficacy in pain relief as well as improvements in sleep and global impressions of changes ( integrating the effect of treatment and side - effects ) , and some studies also in quality of life measures ( lesser et al 2004 ; sabatowski et al 2004 ) and mood ( rosenstock et al 2004 ) . two randomized placebo - controlled trials have been conducted in central pain ( table 2 ) . the first study published is a parallel group design study in central neuropathic pain due to spinal cord injury ( siddall et al 2006 ) . a baseline week was followed by a 3-week titration period where pregabalin was increased up to 300 mg bid and a 9-week fixed dose period . concurrent pain medication was kept constant during the trial and included tricyclic antidepressants in 33% in the pregabalin group and 18% in the placebo group , opioids in 30% and 48% , and antiepileptic drugs except gabapentin in 11% and 9 % respectively . muscle relaxants ( including baclofen ) were used by 54% in the pregabalin group and 37% in the placebo group while benzodiazepines were used by 40% and 39% respectively . pain was evaluated daily on a numeric rating scale ( nrs , 010 ) and the primary efficacy measure was the weekly mean pain score at endpoint ( last week on study drug ) . the improvement in pain score from baseline ( pregabalin placebo ) was 1.53 ( 0.92 to 2.15 ) , similar to values observed in studies in peripheral neuropathic pain . the effect was significant from week 1 and remained so for the duration of the study . the nnt for 50% pain relief ( 7.1 ( 3.937 ) ) was higher than in most peripheral neuropathic pain studies ; however , the nnt for 30% pain relief ( 3.9 ( 2.59.1 ) ) and pain improvement on the patient global impression of change ( 2.9 ( 205.1 ) ) was similar to what is observed in post - herpetic neuralgia and painful diabetic neuropathy ( lesser et al 2004 ; rosenstock et al 2004 ; sabatowski et al 2004 ; freynhagen et al 2005 ; van seventer et al 2006 ) . recently , pregabalin was studied in a parallel group design study in patients with central pain following stroke or spinal cord injury ( vranken et al 2007 ) . the etiology was stroke in 19 patients ( of these thalamic lesion in 4 and brainstem infarction in 3 ) and spinal cord injury in 21 patients ( of these 11 had a complete injury ) . the diagnoses were evenly distributed among patients allocated to pregabalin ( n = 20 ) and to placebo ( n = 20 ) . for the diagnosis of central pain , the pain should be described as burning , paroxysmal episodes of shooting pain , or pain on light touch , and patients had to score above 12 on the leeds assessment of neuropathic symptoms and signs questionnaire ( lanss ) ( bennett 2001 ) . a baseline pain score above 6 ( visual analog scale , vas ) was required . in a flexible - dose regime and with no base - line period , patients received escalating doses of either pregabalin tablets 150 mg or matching placebo capsules bid titrated at 3-day intervals until a pain reduction of 1.8 on a vas was obtained , they reached the maximum daily dose of 600 mg , or had intolerable side - effects . the patients then remained on the final dose during the remainder of the study period , which was 4 weeks . patients treated with gabapentin discontinued this treatment at least 3 days before receiving study medication . pain medication that was continued during the trial was opioids in 53% , antidepressants in 30% , and carbamazepine in 10% . seventeen patients in the pregabalin group completed the study : nine received 600 mg and eight received 300 mg daily . the primary efficacy parameter was the pain intensity based on the average of 3 vas pain scores measured during the 24 hours prior to baseline and at the end of the 4-week treatment period . the improvement in pain score from baseline ( pregabalin placebo ) was 2.18 ( 0.57 to 3.80 ) with no difference in efficacy between the groups with spinal and brain injury . the nnt for 50% pain relief was low , 3.3 ( 1.914.3 ) , and for 30% pain relief it was 4.0 ( 2.0328 ) . in both studies , pregabalin was an add - on analgesic , which suggests that responses may be due to synergistic interactions . in painful diabetic neuropathy pregabalin was effective as monotherapy ( lesser et al 2004 ; rosenstock et al 2004 ; richer et al 2005 ) , and in both studies in central pain , effect sizes were similar regardless of whether patients used any concomitant analgesics ( siddall et al 2004 ; vranken et al 2007 ) . this would suggest that pregabalin is effective as monotherapy also in central pain , but potential synergistic effects need to be studied in appropriately designed studies . pregabalin is generally well tolerated with no contraindications except for known hypersensitivity to pregabalin or its components . the most common adverse reactions in the peripheral neuropathic pain studies were dose - related dizziness ( 22%38% ) and somnolence ( 11%25% ) , which does not resolve in about one third of patients . other adverse reactions were dry mouth , asthenia , blurred vision , ataxia , peripheral edema , and weight gain not limited to patients with edema . pregabalin treatment is not associated with clinical significant withdrawal syndromes ( frampton and foster 2006 ) , but abrupt discontinuation may cause insomnia , nausea , headache , or diarrhea and it is recommended to taper off during at least one week ( pfizer 2007 ) . in case of persistent blurred vision , a visual field testing and funduscopic examination may be considered , and patients are advised to report unexplained muscle pain particularly if accompanied with malaise and fever ( pfizer 2007 ) due to unsettled relation of pregabalin to rhabdomyolysis and creatine kinase elevations . pregabalin is recommended to be used with caution in patients with congestive heart failure ( nyha , ( new york heart association ) class iii and iv ) because of limited data in this population ( pfizer 2007 ) . in the two studies in central pain , the frequency of somnolence in the trial by siddall et al ( 2006 ) ( 41% in the pregabalin group and 9% in the placebo group ) was more common than in studies in peripheral neuropathic pain , which may be attributed to additive effects of concomitant medications such as baclofen and benzodiazepines in this patient population . in the study by vranken et al ( 2007 ) , somnolence occurred in 45% but was equally common in the placebo group . other more frequent adverse reactions in the pregabalin group in the siddall study included : dizziness observed in 24% , edema in 20% , asthenia and dry mouth each in 16% , constipation in 13% , amnesia in 10% , and blurred vision in 9% . the frequency of peripheral edema ( 10% ) was not higher than that observed in peripheral neuropathic pain . two patients in the placebo group and eight in the pregabalin group had a weight gain 7% . the median time to onset of somnolence and dizziness was within 8 and 6 withdrawal due to side - effects occurred in 15 pregabalin- and 9 placebo - treated patients . two adverse reactions were considered related to treatment : one had a withdrawal reaction 1 day following pregabalin discontinuation with increased spasticity and impaired coordination , and one had edema , hypervolemia and reduced platelet count caused by an infection . in the study by vranken et al ( 2007 ) , side - effects were mild to moderate with no difference in frequency of adverse reactions in the two study groups . few other randomized trials have been performed in central pain ( finnerup and sindrup 2007 ) . the related drug gabapentin has been studied in spinal cord injury pain ( levendoglu et al 2004 ) . gabapentin up to 3600 mg relieved intensity and frequency of pain and several pain descriptors in 20 paraplegics with complete spinal cord injury . the tricyclic antidepressant ( tca ) amitriptyline has been studied in a three - way crossover study in post - stroke pain ( leijon and boivie 1989 ) . amitriptyline in doses up to 75 mg daily had a significant pain - relieving effect , which correlated with total plasma concentration . amitriptyline did not relieve nociceptive and neuropathic pain in spinal cord injury , but neuropathic pain was not evaluated separately ( cardenas et al 2002 ) . the anticonvulsant and sodium blocker lamotrigine reduced central post - stroke pain in doses of 200 mg / day as well as cold allodynia ( vestergaard et al 2001 ) , but in spinal cord injury pain , lamotrigine 200400 mg daily was not more effective than placebo in reducing pain , although a post - hoc analysis suggested that it may be effective in a subgroup of patients with incomplete injury and evoked pain ( finnerup et al 2002 ) . in multiple sclerosis , cannabinoids have been shown to relieve central pain ( svendsen et al 2004 ; rog et al 2005 ) . carbamazepine did not relieve post - stroke pain ( leijon and boivie 1989 ) and mexiletine ( chiou - tan et al 1996 ) and valproate ( drewes et al 1994 ) had no significant effect in spinal cord injury pain , but these studies all include a low number of patients with a risk of a type ii error . treatment of central and peripheral neuropathic pain is limited by side - effects and high potential for drug interaction . side - effects to tcas attributed to anticholinergic actions are common , eg , dry mouth , constipation , and urinary retention , and there is a risk of somnolence and confusion , orthostatic hypotension , and gait disturbances . the most serious side effect is cardiotoxicity ( ray et al 2004 ) , and tcas are contraindicated in patients with heart failure and cardiac conduction blocks , and ecg is therefore needed before initiating treatment . lamotrigine treatment is associated with dizziness , ataxia , diplopia , somnolence , nausea , and allergic exanthema and stevens - johnson syndrome , and very slow - dose escalation is recommended . side - effects to cannabinoids include dizziness , drowsiness , impaired psychomotor function , and other psychoactive effects like dysphoria , and there is an unsettled issue with respect to risk of precipitating psychosis or schizophrenia ( semple et al 2005 ) . other side - effects include confusion , especially in elderly patients , urinary retention , dizziness , and dysphoria as well as risk of abuse and addiction . it is therefore recommended to consider long - term opioids for non - cancer pain only when other reasonable therapies fail to provide adequate pain relief ( kalso et al 2003 ) . in summary , amitriptyline ( central post - stroke pain ) , lamotrigine ( central post - stroke pain but a negative trial in spinal cord injury pain ) , gabapentin ( spinal cord injury pain ) , and pregabalin ( spinal cord injury and central post - stroke pain ) , and cannabinoids ( central pain in multiple sclerosis ) have proven to be effective , but large - scale randomized controlled studies are lacking , and a treatment algorithm for central pain still needs to be based partly on established treatments for peripheral neuropathic pain for which tcas , serotonin - noradrenaline reuptake inhibitors , gabapentin / pregabalin , and opioids / tramadol have consistently shown efficacy ( finnerup et al 2005 ) . there is no evidence to suggest that pregabalin and gabapentin have different efficacy or side effects determined by nnt or nnh values ( finnerup et al 2005 ) . differences between the two drugs relates to slightly higher expenses for pregabalin at the moment but more favorable dosing ( twice daily dose possible ) and linear kinetics with pregabalin . thus , based on evidence for efficacy , gabapentin , pregabalin , and tcas consistently relieve peripheral neuropathic pain as well as central pain ( except for the lack of efficacy of amitriptyline in spinal cord injury mixed pain ) . of these , gabapentin and pregabalin may be considered the first - line drugs for the treatment of central pain due to their consistent efficacy , safety , and minimal potential for drug - drug interactions , and tcas a second line - treatment if no contraindications exist . possible third - line treatments for central pain include opioids and tramadol , cannabinoids in multiple sclerosis , lamotrigine , and serotonin - noradrenaline reuptake inhibitors , which have not yet been tested in central pain but have a better side - effect profile than tcas . in many cases , treatment provides no or only partial pain relief , and often combination therapy is used . there is a strong rational for combining drugs with different mode of actions ( backonja et al 2006 ) , but little clinical evidence . in one randomized trial , gabapentin and morphine combined achieved better analgesia at lower doses than with either drug alone ( gilron et al 2005 ) . the recommended dose of pregabalin is 75150 mg twice daily or 50100 mg 3 times a day in patients with a creatinine clearance of at least 60 ml / min . dosing is usually started at 75 mg once or twice daily and may be increased to 300 mg / day within 1 week based on efficacy and tolerability . patients with post - stroke pain may be more susceptible to medication than other patient populations . for that reason it is advisable in some patients to start with a low pregabalin dose of 25 mg and increase slowly . based on efficacy and safety , pregabalin is considered a first - line drug together with gabapentin in the treatment of central pain . pregabalin and gabapentin may especially have a primary role in patients with anxiety and sleep disturbances and in patients who are taking multiple drugs . somnolence , dizziness , and accidental falls may be a concern , especially in the elderly and in those treated with other drugs with cns - related side - effects .

rnaomics , analogous to proteomics , concerns aspects of the secondary and tertiary structure , folding pathway , kinetics , comparison , function and regulation of all rna in a living organism . given recently discovered roles played by micro rna , small interfering rna , riboswitches , ribozymes , etc . , it is important to gain insight into the folding process of rna sequences . we describe the web server rnaloss , which provides information about the distribution of locally optimal secondary structures , that possibly form kinetic traps in the folding process . the tool rnaloss may be useful in designing rna sequences which not only have low folding energy , but whose distribution of locally optimal secondary structures would suggest rapid and robust folding . website : . central neuropathic pain ( central pain ) is treated with antidepressants , various anticonvulsants , opioids , and cannabinoids , but in many cases treatment is insufficient and associated with a range of side - effects . this review addresses a new treatment for neuropathic pain , the anticonvulsant pregabalin . we review the pharmacology , mode of action , pharmacokinetics , and safety of pregabalin as well as two randomized efficacy studies in central pain and a brief overview of efficacy in peripheral neuropathic pain . pregabalin appears to have efficacy in treating central pain comparable to that in peripheral neuropathic pain as well as efficacy of other recommended drugs for central pain . pregabalin also improves disturbed sleep and anxiety . pregabalin is well tolerated ; the most common side - effects are somnolence , dizziness , ataxia , and weight gain . pregabalin is suitable for patients on multiple drugs although there may be additive cns - related side - effects . thus , pregabalin has a primary role in central pain patients .

toxic cyanobacterial blooms in eutrophic lakes , ponds , and reservoirs are a common occurrence around the world [ 13 ] . cyanobacteria of the genera microcystis , anabaena , nostoc , and planktothrix produces a wide range of potent toxins , including a family of heptapeptide hepatotoxins , referred to as microcystins ( mcs ) . microcystins are the most frequently detected cyanobacterial toxins , which cause hepatotoxicity and tumor promotion in wild animals , livestock , and humans [ 2 , 4 , 5 ] . epidemiological studies of primary liver cancer in china and the death of 56 patients during a dialysis treatment in caruaru , brazil triggered worldwide concern about toxicity of mcs [ 4 , 5 ] . approximately 75 variants of mcs have been identified and mc - l ( leucine ) r ( arginine ) is the most common variant of mc worldwide [ 1 , 69 ] . limited studies in new zealand ( nz ) have reported the occurrence of the [ dha]mc - lr variant occurring with high frequency in nz waters [ 3 , 10 , 11 ] . microcystins are chemically stable over a wide range of temperature and ph , possibly as a result of their cyclic structure . the toxins are also resistant to enzymatic hydrolysis by some general proteases , such as pepsin , trypsin , collagenase and chymotrypsin . however , in the presence of natural microbial populations , degradation of mcs can take place . in 1994 , jones et al . successfully isolated from australian river water sphingomonas strain mj - pv ( acm-3962 ) , a single bacterial strain that utilized mc - lr as its sole source of carbon , and nitrogen needed for growth . further research led to the elucidation of mc - lr degradation pathways of the bacterial strain acm-3962 . two intermediate degradation products were identified , suggesting that at least three intracellular hydrolytic enzymes were involved in the degradation of mc - lr . the first enzyme identified in the degradation pathway , a metalloprotease named microcystinase , cleaves the aromatic ring of mc - lr at the arg - adda peptide bond . this step yields a linearized mc - lr , which has a 160-fold reduction in toxic activity compared with the parent mc - lr . next , a serine peptidase catalyzes the linearized mc - lr at the ala - leu peptide bond , producing a tetrapeptide . finally , the third enzyme , another metalloprotease , cuts the peptide bonds randomly resulting in undetectable peptide fragments and amino acids . further performed cloning and molecular characterization of four genes ( mlra , b , c , and d ) that encode the three hydrolytic enzymes plus a putative oligopeptide transporter , all involved in the mc degradation metabolic pathway of the sphingomonas strain acm-3962 . cyanobacterial blooms and mc production in nz water bodies have been investigated by wood who detected mcs in 102 water samples from 54 different locations . the samples collected from lake rotoiti , lake hakanoa , lake horowhenua , and lake waitawa in april 2003 contained high levels of mcs , ranging from 0.02 g ml to a maximum of 36,500 g ml . wood revealed the presence of [ dha]mc - lr , mc - lr , mc - rr , mc - ar , mc - fr , mc - la , mc - wr , mc - yr , and mc - ly from lake horowhenua , and mc - lr , mc - rr , mc - ar , mc - fr , mc - la , mc - wr , and mc - yr from lake rotoiti . somdee purified seven mc variants from lyophilized bloom samples ( 20 g each ) of microcystis aeruginosa collected from lake horowhenua in may 2005 , with [ dha]mc - lr , the major variant ( purity of 93% and total yield of 51.84 mg ) , along with moderate amounts of mc - lr , mc - rr , mc - dme - rr , mc - ar , mc - fr , and mc - yr . contamination of drinking water sources by cyanotoxins remains a serious threat to animal and human health and thus degradation of mcs by naturally occurring bacteria is an attractive bioremediation option for removing mcs from drinking and recreational water sources and supplies . the objectives of this research were to ( a ) isolate and identify natural aquatic bacteria from nz water bodies capable of degrading mcs , ( b ) ascertain optimal biodegradation conditions for the identified nz mc - degrading bacteria , and ( c ) determine the biodegradation pathway by the identified nz mc - degrading bacteria for [ dha]mc - lr , the most common mc variant in nz waters . water samples taken from lake rotoiti , lake rotorua , lake rotoehu , and lake horowhenua , during blooms of microcystis aeruginosa , were used as potential sources of mc - degrading bacteria . 10 ml samples of lake water were inoculated into 190 ml of a sterile mineral salts medium ( msm ) broth containing [ dha]mc - lr and mc - lr as the main food source for 5 days and incubated in the dark at 30c in a shaking incubator ( 200 rpm ) . mc - degrading bacteria were isolated by streaking serial dilutions of 10 to 10 onto peptone - yeast extract medium agar plates ( pyem ) and incubated at 30c . a single colony of each bacterial isolate was grown on 5 ml pyem broth overnight at 30c in a shaking incubator ( 200 rpm ) . 1 ml of overnight culture was inoculated into 19 ml of fresh msm broth containing mcs at a final concentration of 25 g ml and incubated at 30c in a shaking incubator ( 200 rpm ) for 7 days . a 1 ml sample was withdrawn at 1 day intervals , centrifuged ( 12000 rpm for 10 min ) , and the supernatant analyzed on hplc ( uv detector ) . sequencing of 16s rrna was carried out by esr , new zealand , using an abi prism bigdye terminator dna sequencing kit and analyzed on a model 3730xl abi dna sequencer ( applied biosystems ) . the dna sequences were compared with the genbank , embl , and djb prokaryote databases using the default settings of the fasta3 alignment programme through the ebi server . a mix of [ dha]mc - lr and mc - lr was considered ideal as a substrate for the biodegradation experiments ( determination of optimal degradation conditions and examination of biodegradative by - products ) , as extracts rich in [ dha]mc - lr have not previously been studied , and new zealand natural bloom sources were deemed more appropriate than pure commercial forms of mc . extraction and purification of [ dha]mc - lr and mc - lr mcs from lyophilized bloom samples were performed as described by somdee . the pure bacterial isolate nv-3 was cultured in a pyem broth for 36 h ( late exponential growth phase determined from bacterial growth curve experiment ) in a shaking incubator at 30c and 200 rpm . the bacterium was centrifuged at 12,000 rpm for 5 min ( 4c ) . the supernatant was decanted , and the pellet was resuspended in 0.05 m phosphate buffer , ph 7.0 . the final pellet was resuspended in 5 ml of sterile msm broth , and this bacterial culture was used for examining the effect of temperature , bacterial , and mc concentrations on mc degradation by the bacterium isolate nv-3 . sterile msm broth was added to the prepared bacterial culture to adjust the concentration of the stock culture to give an optical density of 1.0 ( od600 ) and bacterial concentration of approximately 1.0 10 cfu / ml . to establish the effect of temperature on mc degradation , 9 ml of the nv-3 bacterial culture was mixed with 1 ml of [ dha]mc - lr and mc - lr ( 25 g / ml final concentration ) . the experiment was carried out in triplicate at 6 different temperatures , 10 , 15 , 20 , 25 , 30 , and 35c , in a shaking incubator at 200 rpm . the biodegradation of mcs was monitored for each experiment over a period of 28 days . an aliquot ( 1 ml ) of the bacterial / cyanotoxin mix was withdrawn after 0 , 1 , 3 , 5 , 7 , 10 , 14 , 21 , and 28 days of incubation and centrifuged at 12,000 rpm for 10 min . the mc concentration was determined in the supernatant using hplc . using the optimum temperature for mc degradation , experiments to determine the effect of bacterial concentration on mc degradation were carried out at different bacterial concentrations with a fixed mc concentration of 25 g / ml . five bacterial concentrations were prepared od600 = 0.1 ( bacterial concentration approximately 7.9 10 cfu / ml ) , od600 = 0.3 ( 2.5 10 cfu / ml ) , od600 = 0.5 ( 4.9 10 cfu / ml ) , od600 = 1.0 ( 1.0 10cfu / ml ) , and od600 = 1.5 ( 1.45 10 cfu / ml ) , using the prepared bacterial culture and adding sterile msm broth until the required od at 600 nm was obtained . a 9 ml sample of each bacterial concentration was mixed with 1 ml of [ dha]mc - lr and mc - lr ( 25 g / ml final concentration ) . the experiments were carried out in a shaking incubator at the optimum temperature ( 30c ) and at 200 rpm . the optimum temperature and bacterial concentration were used to establish optimum mc concentration for bacterial degradation . 9 ml of the bacterial culture ( od600 = 1.0 ) was mixed with 1 ml of [ dha]mc - lr and mc - lr , yielding final mc concentrations of 1 , 10 , 25 , and 50 g / ml , and incubated in a shaking incubator at 30c and 200 rpm . the [ dha]mc - lr and mc - lr variants extracted and purified from natural algal bloom were used in the biodegradation assays for detection of mc - degradation by - products by the nv-3 isolate . the pure nv-3 isolate was transferred to 19 ml of fresh msm broth , containing mcs at a final concentration of 25 g ml and incubated in a shaking incubator at 30c and 200 rpm . samples ( 1 ml ) were taken every 6 h until 48 h , and the mc - degradation by - products were detected and analyzed using lc / ms - ms at cawthron institute , nelson , new zealand . the isolated bacterium ( nv-3 isolate ) was cultured in peptone - yeast extract broth for 36 h at 30c and 200 rpm . genomic dna was extracted using the wizard genomic dna purification kit ( promega ) and quantified by a biophotometer ( eppendorf ) with a260/280 ratio . and for mlrb , mlrc , and mlrd genes are described in ho et al . . the pcr reactions were composed of 1 ng genomic dna , 1 pmol of each primer , 1 pcr buffer ( invitrogen ) , 2 mm dntps ( invitrogen ) , 2.5 mm mgcl2 ( invitrogen ) , and 1.25 units of amplitaq gold dna polymerase ( applied biosystems ) , giving a final volume of 20 l . the amplification was performed on a programmable thermal cycler ( hybaid p2 thermal cycler ) with temperatures and times as previously described by saito et al . for mlra and were then purified using the wizard sv gel and pcr clean - up system ( promega ) , and both strands were directly sequenced on an abi-3730 automated sequencer ( applied biosystems ) , at allan wilson centre ( awc ) genome sequencing centre , massey university , nz . assembled sequences were aligned using a clustalw with mega 4.0 program ( retrieved december 10 , 2008 , from http://www.megasoftware.net/ ) and then subjected to a nucleotide blast search . water samples obtained from lake rotoiti , lake rotoehu , lake rotorua , and lake horowhenua were plated on msm broth with [ dha]mc - lr and mc - lr as the sole carbon and nitrogen source . a total of 27 isolates of different types , shapes , and colors of colony were selected ; however , only three isolates that were obtained from lake rotoiti , designated nv-1 , nv-2 and nv-3 , were truly able to break , down the mcs ( data not shown ) . nv-3 isolate showed greatest degradation activity and was used for characterization and further experiments . the 16s rrna sequence of the isolate nv-3 was determined and compared with the genbank , embl , and djb prokaryote databases . the databases revealed that the 16s rrna sequences of the nv-3 isolate resemble the sequences of sphingomonas strain md-1 ( ab110635 ) , with 100% sequence homology for 1436 continuous nucleotides , and exhibits 98.5% homology with 16s rrna of sphingomonas stygia ( ab025013 ) . the nv-3 isolate was classified as a sphingomonas sp . , indistinguishable from the sphingomonas strain md1 . the effect of temperature on the ability of nv-3 ( 1.0 10 cfu / ml ) to degrade 25 g / ml mcs was investigated . mc degradation by nv-3 began on day 1 under all temperatures tested ( figure 1(a ) ) ; however , the rate of mc degradation varied with temperature . the degradation rate was slowest at 10c ( 0.89 g / ml / day ) and steadily increased with increasing temperatures to 35c ( 8.30 g / ml / day ) . mc concentrations rapidly decreased at temperatures from 20 to 35c , with complete degradation occurring within 5 days ( figure 1(a ) ) . however , at these temperatures a noticeable drop off in degradation rate occurred after the initial rapid phase . the highest degradation rate was reached with temperatures of 30c ( 8.33 g / ml / day ) ; however , the rate of degradation during the initial rapid phase was similar between temperatures of 25 , 30 , and 35c and was calculated to be approximately 8.30 g / ml / day . these experiments demonstrated that the optimum temperature for nv-3 isolate biodegradation of [ dha]mc - lr and mc - lr variants was 30c . the optimum bacterial concentration of nv-3 isolate for degradation of 25 g / ml mcs was then investigated at 30c ( being the optimum temperature for mc degradation by nv-3 ) . bacterial concentrations of 7.9 10 , 2.5 10 , 1.0 10 , and 1.45 10 cfu / ml completely degraded the mcs within 3 days ( figure 1(b ) ) . however , the degradation rate was slow with the lowest bacterial concentrations 7.9 10 and 2.5 10 cfu / ml . after one day of incubation with 7.9 10 and 2.5 10 cfu / ml nv-3 isolate , the amount of [ dha]mc - lr remaining was 80% and 40% , respectively . by contrast , after incubation with the higher nv-3 isolate concentrations of 4.9 10 , 1.0 10 , and 1.45 10 cfu / ml , less than 20% of [ dha]mc - lr remained . the mc degradation rate in general increased with increasing bacterial concentration . at bacterial concentrations of 4.9 10 , 1.0 10 and 1.45 10 cfu / ml , the degradation rates were not significantly different with the same degradation rate of 8.33 g / ml / day . however , the degradation rate actually decreased slightly at the highest bacterial concentration of 1.45 10 cfu / ml . these experiments demonstrated that the optimum bacterial concentration for nv-3 isolate biodegradation of [ dha]mc - lr and mc - lr variants was 1.0 10 cfu / ml . the effect of variation in mc concentration on nv-3 isolate degradation capability was also investigated at 30c and nv-3 concentration 1.0 10 cfu / ml . at low mc concentrations ( 1 and10 g / ml ) , the toxins were completely degraded in one day , whereas at higher concentrations of toxins ( 25 and 50 g / ml ) degradation took longer , reaching undetectable levels by day 3 and 6 respectively ( figure 1(c ) ) . the rate of degradation for concentrations of 25 and 50 g / ml was equal at 8.33 g / ml / day . biodegradation of mcs was induced using a mixture of [ dha]mc - lr and mc - lr ( 25 g / ml final concentration ) with cell suspensions of the isolate nv-3 ( 1.0 10 cfu / ml ) . the toxin concentration began to decline , and two peaks , referred to as by - products a and b , were detected in a hplc chromatogram . the peaks corresponding to the by - products increased gradually , while the peak corresponding to the parent toxins decreased . the by - products from microbial catabolism were further analyzed using lc / ms - ms . [ dha]mc - lr is 14 mass units less than mc - lr due to the loss of a methyl group at mdha , and the m / z at 981.75 and 995.75 confirmed that [ dha]mc - lr , and mc - lr , respectively , were present in the biodegradation assay samples ( figures 2(a ) and 2(b ) ) . by - product a revealed the base peak at m / z 999 ( figure 2(c ) ) and was analogous to the linearized peptide of [ dha]mc - lr ( nh2-adda - d - glu - dha - d - ala - l - leu - d - measp - l - arg - oh ) . the ion spectra of the prominent ion at m / z 848.4 ( figure 2(c ) ) , which was from the loss of phch2chome from the linearized peptide of [ dha]mc - lr , confirmed the ring opening of [ dha]mc - lr . the biodegradation by - product b revealed the base peak at m / z 601.2 ( figure 2(d ) ) . the [ m+h ] ion 601.2 is the tetrapeptide of [ dha]mc - lr ( nh2-adda - d - glu - dha - d - ala - oh ) , 14 mass units less than the tetrapeptide of mc - lr microbial degradation reported in bourne et al . . dna fragments of the nv-3 isolate mlra ( 720 bp ) , b ( 340 bp ) , c ( 590 bp ) , and d ( 600 bp ) genes were pcr amplified and then directly sequenced . the dna sequences were investigated using blastn search . the mlra nucleotides ( 721 bp ) exhibited a 99% dna sequence similarity to the 807 base pair nucleotide sequence of the mlra gene from sphingomonas strain md-1 ( ncbi accession number ab114202 ) . the mlrb337 bp revealed a 94% similarity to the 448 nucleotide base pair sequence of the mlrb gene from sphingopyxis sp . lh21 ( dq423530 ) , whereas the nucleotide sequence of mlrc ( 588 bp ) and mlrd ( 597 bp ) genes was 99% and 97% similar to the respective genes ( with 666 bp and 671 bp ) from sphingomonas sp . the presence of microcystins ( mcs ) in freshwater is becoming an increasing problem and poses a potential threat to human health around the world . the mc toxin is well recognized as a stable and persistent compound ; however , reports have shown that mcs are vulnerable to break down by indigenous bacteria established in natural water [ 2 , 14 , 2225 ] . this is the first study to isolate and characterize indigenous mc - degrading bacteria in new zealand waters . we have demonstrated that the bacterium isolate nv-3 from lake rotoiti is able to utilise [ dha]mc - lr and mc - lr as a sole source of carbon and energy . on the basis of 16s rrna sequences the isolate nv-3 is indistinguishable from the sphingomonas strain md-1 from japan . the rate of mc degradation by any bacterial isolate , including nv-3 , relies mainly on the incubation temperature as well as bacterial concentration . biodegradation of mcs by the bacterium occurred under a wide range of temperatures between 10c and 35c . at 10c , the biodegradation was very slow and identical to that of the mc degrading - bacterium sphingopyxis strain lh21 , isolated from australia , suggesting that it would similarly not be able to degrade mcs at 4c . the ability of bacteria to degrade toxins in low temperatures is relevant to the degradation of toxins in winter , for example , when the water temperature is low . for nv-3 , increases in water temperature from 15c to 30c were associated with an increase in biodegradation rate ; however , temperatures higher than the optimum temperature ( 30c ) for nv-3 bacterial growth resulted in slightly decreased degradative ability . it is likely that at high temperatures , nv-3 bacterial cells are unable to produce mc - degrading enzymes , or the cells might be inactive or growing slowly . the highest rate of mc - degradation by the isolate nv-3 was achieved at 30c , a similar temperature observed for maximum mc - degradation by the sphingomonas strain y2 , isolated from lake suwa , japan , and for sphingopyxis strain tt25 isolated from australian waters , the highest rate was achieved at 25c . at the optimum temperature of bacterial growth , the bacterial metabolism is very active , producing a lot of enzymes responsible for mc degradation . by contrast , at temperatures that are lower or higher than that for optimum growth , bacterial metabolism and the production of biodegradation enzymes are presumably less active , slowing the rate of mc degradation . the degradation rate increased with increasing bacterial cell concentration ; however , concentrations above 1.0 10 cfu / ml resulted in a decrease in degradative ability . this decrease might be due to competition or inhibition between the cells at very high cell numbers , and , therefore , the optimum cell density is also crucial for toxin degradation . in this study , the optimum cell density of the bacterium isolate nv-3 , required for mc degradation experiments , was between 4.9 10 to 1.0 10 cfu / ml and the minimum number of cells required was approximately 7.9 10 cfu / ml . it is interesting that at the minimum cell density , mcs were completely degraded within 3 days , the same length of time taken with higher concentrations of bacteria . this confirms the ability of the bacterium isolate nv-3 to utilise the toxins as their own food and to multiply itself to a cell density sufficient to completely degrade the mcs . the effect of toxin concentrations on mc degradation by nv-3 was also examined . in this study , at the optimum temperature ( 30c ) and bacterial concentration ( 1.0 10 cfu / ml ) , the biodegradation rate increased with increasing concentrations of the toxins ( the mixture of [ dha]mc - lr , and mc - lr ) . the degradation rate by the bacterial isolate was identical ( i.e. , 8.33 g / ml / day ) for moderate to high concentrations of the toxins ( 25 g / ml to 50 g / ml ) . it is possible that as the sole carbon and nitrogen sources for bacterial growth , high concentrations of mcs were beneficial to growth of the isolated bacteria . however , the apparent inhibitory effects of very high toxin concentrations ( i.e. , > 50 g / ml ) and the limit to the amount of mcs that nv-3 can degrade need to be further investigated , as the effects on bacterial growth and metabolism are unknown . biodegradation studies of mcs have been mainly focused on the mc - lr variant since it is found worldwide and possesses high toxicity ( ld50 ( ip ) = 50 g / kg in mice ) [ 1 , 8 , 9 , 28 ] . other analogues of mcs such as mc - rr , yr , lw , and lf have also been examined [ 13 , 26 , 2935 ] . in new zealand waters it appears that [ dha]mc - lr is in greater quantity than mc - lr [ 3 , 10 , 11 ] . in this study , in addition to mc - lr , [ dha]mc - lr was used as a substrate to characterize the biodegradation pathway of [ dha]mc - lr using the bacteriumisolate nv-3 . previous studies have shown that at least three hydrolytic enzymes are involved in degradation of mc - lr [ 15 , 16 ] . our study has shown that the by - products a and b of [ dha]mc - lr ( figures 2(c ) and 2(d ) ) are degradation products equivalent to what happens to mc - lr ( figure 3 ) in an enzymatic pathway identical to that described by bourne et al . . it is important to note that the mc - degradation by - products , namely linearized peptides , tetrapeptides and peptide fragments are significantly less toxic than the parent molecules of mcs . demonstrated that the toxicity of the linearized peptides of mc - lr is reduced 160-fold compared with the parent compound , while other studies have demonstrated that tetrapeptide and amino acids are nontoxic . these findings strongly indicate that microbial degradation is a potentially safe and natural treatment for removing mcs from water . in summary , ( 1 ) bacteria were found in new zealand lakes with the ability to degrade [ dha]mc - lr and mc - lr as the sole carbon and nitrogen sources ; ( 2 ) 16s rna of the mc - degrading bacteria isolated were indistinguishable from a previously identified bacterium md-1 which was located in japan ; ( 3 ) optimal nv-3 degradation of 25 g / ml [ dha]mc - lr and mc - lr prepared form a natural algal bloom , occurred at 30c with bacterial concentration of 1.0 10 cfu / ml , and(4 ) the by - products a and b from biodegradation of [ dha]mc - lr and the detection of mlra , mlrb , mlrc , and mlrd genes in nv-3 genome indicate that degradation of [ dha]mc - lr is via a similar mechanism for degradation of mc - lr as described by bourne et al . [ 15 , 16 ] . this study has demonstrated that microcystin - degrading bacteria are present in new zealand water bodies , and that these bacteria could be used potentially on a larger scale for removing microcystins from water . the four gene sequences mlra , mlrb , mlrc , and mlrd obtained for sphingomonas nv-3 isolate have been deposited in genbank under accession numbers jn256930 , jn256929 , jn256928 , and jn256927 , respectively . pancreatic heterotopia is defined as the presence , outside its usual location , of pancreatic tissue which lacks anatomical and vascular continuity with the pancreas proper ( 1 ) . the heterotopic pancreas ( hp ) is a relatively uncommon congenital anomaly , with an incidence between 0.55% and 13.7% in autopsy series and mean frequency between 1 and 2% . hp has been found in all age groups , predominantly in the sixth decade of life ( 2 ) . the usual locations of hp are in the stomach in 25 - 38% cases , the duodenum in 17 - 36% and the jejunum in 15 - 22% of cases . it is usually silent but it may become clinically evident when complicated by inflammation , bleeding , obstruction or malignant transformation ( 3 ) . symptomatic patients require surgical exploration in order to obtain a definitive diagnosis and to exclude malignancy . a 12 years old male child presented with severe abdominal pain and intermittent vomiting , not relieved with medications . usg showed telescopy of gut loop along with its mesentry into other infraumblical region at the level of anterior superior iliac spine suggestive of ileoileal intesusception . an ileal polyp was found to be the cause of intussusceptions which was removed along with small segment of adjacent bowel and sent for histopathological examination . surgical specimen of resected ileal polyp with adjacent bowel loop ( 1a ) cut surface of which was pale yellow ( 1b ) . histological examination revealed presence of pancreatic tissue in muscularis propria of ileum ( 1c ) ( h&e , x40 ) with overlying mucosa showing congestion and metaplasia ( 1d ) ( h&e , x40 ) on gross examination , the polyp was brown , oval sessile mass with a broad base measuring 63.52 cm ( fig . the histological examination revealed the presence of pancreatic tissue in the muscularis propria of ileum ( fig . 1c ) made up of pancreatic acini and dialated ducts interspersed by smooth muscle bundles ( fig . photomicrograph showing pancreatic acini and ducts ( h&e , x100 ) the patient had an uneventful recovery and remains asymptomatic postoperatively . as stated by hunt and bonesteel ( 5 ) the first case of heterotopic pancreas was reported by schultz in 1729 , and klob provided its histological confirmation in 1859 ( 6 ) . the reported incidence in autopsy studies is 0.5 - 13% ( 3 ) . in adults it is found mainly in the stomach , duodenum and jejunum , in much smaller proportions in the ileum and meckel s diverticulum , and it is rarely found in the esophagus , liver , gallbladder , omentum , lungs , mediastinum , fallopian tubes and umbilicus ( 2 ) . in adults the incidence is higher in males , while in pediatric patients the female gender prevails . the proposed theory is that during rotation of foregut in a fetus and fusion of dorsal and ventral parts of pancreas , small islands of pancreas are carried away and continue to develop at its aberrant location ( 2 ) . most patients with ectopic pancreas are asymptomatic and diagnosis is usually performed during radiological examination or endoscopy of the digestive tract or during surgical explorations motivated by other diseases ( 2 ) . when symptomatic , about 30% of total mimic clinical symptoms similar to diseases that affect the organ in which the heterotopia is located ( 3 ) . usually they present in the form of small yellowish nodules , ranging from 1 mm to 5 cm , typically covered by intact mucosa , and often exhibit a central hole representing exteriorization of the rudimentary pancreatic duct . however , lesions smaller than 1.5 cm do not usually show such an orifice ( 8) . the ectopic pancreatic tissue is detected more frequently in the submucosa and muscularis propria layers of the gastrointestinal tract and may be observed in the sub - serosa or even in the serosa of the affected segment ( 2 ) . the heinrich classification system is frequently used to classify heterotopic pancreas : type 1 ( containing acini , islets and ducts ) , type 2 ( acini and ducts , no islets ) and type 3 ( ducts alone ) ( 9 ) . the preoperative imaging studies ( ultrasonography , endoscopic ultrasonography and computerized tomography ) are not very specific ( 3 ) . hence , in the majority of cases , the diagnosis is made by histological evaluation following resection of a symptomatic or suspicious lesion ( 10 ) . the management of asymptomatic , histologically verified heterotopic pancreas or those found incidentally during other surgery is under debate . although , in the majority of the cases of heterotopic pancreas reported surgical resections were done ; endoscopic mucosal removal can be an attractive , less invasive option for the resection of accessible lesions ( 3 ) . although there have been studies describing clinicopathological analysis of patients presenting with heterotopic pancreas(2,10 ) , ileal heterotopic pancreas has been rarely reported in children . heterotopic pancreas is a rare congenital lesion , often diagnosed incidentally on histopathological examination and should be considered in the differential diagnosis of intestinal mass lesions .

for the first time a microcystin - degrading bacterium ( nv-3 isolate ) has been isolated and characterized from a nz lake . cyanobacterial blooms in new zealand ( nz ) waters contain microcystin ( mc ) hepatotoxins at concentrations which are a risk to animal and human health . degradation of mcs by naturally occurring bacteria is an attractive bioremediation option for removing mcs from drinking and recreational water sources . the nv-3 isolate was identified by 16s rrna sequence analysis and found to have 100% nucleotide sequence homology with the sphingomonas mc - degrading bacterial strain md-1 from japan . the nv-3 isolate ( concentration of 1.0 108 cfu / ml ) at 30c degraded a mixture of [ dha7]mc - lr and mc - lr ( concentration 25 g / ml ) at a maximum rate of 8.33 g / ml / day . the intermediate by - products of [ dha7]mc - lr degradation were detected and similar to mc - lr degradation by - products . the presence of three genes ( mlra , mlrb , and mlrc ) , that encode three enzymes involved in the degradation of mc - lr , were identified in the nv-3 isolate . this study confirmed that degradation of [ dha7]mc - lr by the sphingomonas isolate nv-3 occurred by a similar mechanism previously described for mc - lr by sphingomonas strain mj - pv ( acm-3962 ) . this has important implications for potential bioremediation of toxic blooms containing a variety of mcs in nz waters . heterotopic , aberrant or ectopic pancreas is defined as the presence of pancreatic tissue in topographic anomaly , with no anatomical , neural or vascular connection to the normal pancreas . it is a rare condition found mainly in stomach , duodenum and jejunum . ileal heterotopic pancreas is an uncommon condition and has been rarely reported in children so far . hereby we report a case of heterotopic pancreas presenting as ileal poyp leading to ileoileal intussusception in a 12 year child .

cytomegalovirus ( cmv ) infection still remains a major cause of morbidity and mortality in allogeneic stem cell transplantation ( sct ) recipients . while cmv infection of the central nervous system ( cns ) in acquired immune deficiency syndrome patients has been reported relatively frequently , it is an unusual presentation in allogeneic sct recipients , but fatal in all cases ( 1 - 4 ) . recently , unrelated cord blood or t - cell depleted grafts have been increasingly used as an alternative source of hematopoietic stem cells . however , the use of these grafts has been associated with an increased frequency of unusual cmv infections . here , we report a case of cmv ventriculoencephalitis after unrelated double cord blood sct with an alemtuzumab - containing preparative regimen for philadelphia - positive acute lymphoblastic leukemia . the patient had recurrent cmv dnaemia despite long - term treatment with antiviral agents ( foscarnet combined with ganciclovir ) and anti - cmv immunoglobulin . a 20-yr - old man underwent unrelated cord blood sct using two cord blood units to treat philadelphia - positive acute lymphoblastic leukemia . the patient was treated with total body irradiation ( 1,200 cgy ) , fludarabine ( 150 mg / m ) , cytarabine ( 9 g / m ) , and alemtuzumab ( 20 mg ) as a preparative regimen . for prophylaxis of cmv reactivation , acyclovir ( 10 mg / kg intravenously every 8 hr ) and anti - cmv immunoglobulin ( 150 mg / kg intravenously biweekly ) were given from day -7 until engraftment . the patients achieved successful neutrophil and platelet engraftment on day 27 and 51 , respectively . on day 33 , cmv dnaemia was detected by real - time quantitative polymerase chain reaction ( rq - pcr ) as 3,775 copies / ml and the viral load increased steadily thereafter . the patient was treated preemptively for cmv dnaemia with foscarnet ( 60 mg / kg intravenously every 12 hr for 7 days , followed by 90 mg / kg intravenously daily ) from day 39 to day 93 . during this period , foscarnet had been chosen , instead of ganciclovir , since it had less potential bone marrow toxicity . thirteen days after discontinuation of the foscarnet , cmv retinitis developed with an increase in cmv dna titer . the foscarnet ( 90 mg / kg intravenously every 12 hr ) and anti - cmv immunoglobulin ( 150 mg / kg intravenously biweekly ) were re - administered . however , 7 days later , the cmv rq - pcr titers increased despite the treatment . then we added ganciclovir ( 5 mg / kg intravenously daily ) on top of foscarnet ( 90 mg / kg intravenously daily ) . clinically , the cmv retinitis improved and the cmv dnaemia resolved 40 days after the combination therapy . on day 171 , he was re - admitted complaining of progressive weakness of all four extremities . the peripheral blood showed pancytopenia without leukemic cells ; a wbc count of 1,160/l , hemoglobin of 6.9 g / dl , and a platelet count of 6,000/l . despite administration of foscarnet and 1 ) showed multiple nodularity with high signal intensities in the bilateral fronto - parietal subcortical white matter , periventricular white matter , and the basal ganglia in the t2-weighted and flair images . diffuse high signal changes in the t2-weighted and flair images were also noted along the wall of the lateral ventricles , which were enhanced with gadolinium on the t1-weighted images . lumbar puncture was performed on day 186 ; the results showed a protein concentrate of 66.5 mg / dl , a glucose concentrate of 62 mg / dl ( serum glucose concentrate , 145 mg / dl ) , no white blood cells , and no red blood cells . the microbiology of the cerebrospinal fluid ( csf ) was negative for bacteria , mycobacterium species , and fungi . there was no skin lesion that suggested a varicella zoster virus infection , and the pcr analysis of the csf for herpes simplex virus ( type 1 & 2 ) and human herpes virus 6 were all negative . 2 ) . based on the typical radiologic findings , the positive pcr for cmv from the csf , and recurrent cmv dnaemia in the peripheral blood in spite of long - term antiviral therapy , recently , posttransplant immune reconstitution after sct varies because of availability of alternative donor and variety of preparative regimens and graft manipulations . the unrelated cord blood sct and haploidentical transplantation themselves may be correlated with a high incidence of cmv reactivation , when compared to matched sibling or unrelated sct ( 5 , 6 ) , as well as the use of antithymocyte globulin , fludarabine , and alemtuzumab ( humanized monoclonal cd52 antibody ) as part of the preparative regimen ( 7 - 13 ) . in particular , the combination of fludarabine and alemtuzumab might cause a higher risk of cmv infection and earlier cmv infection following allogeneic sct compared to other preparations ( 8) . ( 7 ) suggested that the patients receiving alemtuzumab , a total dose of 100 mg , had a higher incidence of cmv infection ; a lower dose of alemtuzumab might be related with a reduced risk of cmv infection . our patient received a much lower dose of alemtuzumab , a total dose of 20 mg . however , he had a recurrent cmv reactivation and finally developed cmv retinitis and ventriculoencephalitis . a combination of intravenous foscarnet and ganciclovir has been advocated for treatment of single drug - resistant cmv disease ( 14 , 15 ) . we tried the combination regimen of foscarnet and ganciclovir , expected to have synergistic effects , for the treatment of cmv retinitis because of two reasons ; 1 ) we used the combination of fludarabine and alemtuzumab for cbt preparation , which might have made the patient highly susceptible to cmv reactivation ; 2 ) cmv retinitis developed after treatment with cmv dnaemia despite the recent prolonged treatment with foscarnet . clinically , the treatment failure of cmv disease with antiviral drugs may be associated with antiviral resistance and/or inadequate penetration of the drug into the infected tissue ( 1 , 3 , 16 , 17 ) . the recurrent cmv dnaemia and progressive cmv disease unresponsive to antiviral agents and anti - cmv immunoglobulin for about 150 days , suggest antiviral resistant - mutant cmv in the case . however , it is the limitation of the current study that we could not perform drug susceptibility analysis , cmv genotyping or phenotyping for drug resistance - associated mutations , or measurement of the drug levels in the plasma and csf . in summary , more aggressive prophylaxis as well as preemptive therapy for cmv infection and more diligent monitoring for progression to cmv diseases should be considered for a certain proportion of sct recipients with multiple risk factors of delayed immune reconstitution . lateral gene transfer ( lgt ) is a phenomenon central to prokaryotic evolution and adaptation . with the increasing use and sophistication of genomics technologies and bioinformatics tools , estimates of the extent of laterally transferred genes a recent study estimated that an average of 81% of the genes in any given prokaryote had been transferred at some point in their history . the existence and extent of lgt has long been a vexing issue in biology . in respect of microbiology it has led to a long debate of the species concept as it applies to prokaryotes . in the broader evolutionary debate lgt has also challenged aspects of neo darwinism especially in regards to the notion of evolution by gradual change . while all microbiologists accept lgt as a fact , there are still difficulties in reconciling the observation of its large contribution to genomes in the long - term and the likely fitness impact on a genome of acquiring one or more gene units of dna in the short - term . with as little as 20% of a bacterial genome being comprised of genes that respect the classical evolutionary laws of inheritance by vertical descent it follows that most laterally acquired genes must be integrated into essential interconnected metabolic or regulatory pathways ( cell networks ) . neo - darwinism argues that evolution of genes integrated into cell networks occurs by mutational changes in existing genes or duplicated genes either of which randomly generates subtle fitness advantages . over time these can lead to networks with distinct properties in different species and , indeed , facilitate the process of speciation itself . in contrast , the sudden introduction of a new gene or genes into a bacterium is an instantaneous in contrast to a subtle point mutation in an existing or duplicated gene , this is much less likely to provide an opportunity for the new gene to co evolve with , and adapt to , existing cell networks . in the absence of other changes a partial explanation for this dilemma may be in the finding that mobile dna might be silenced by host defenses preventing detrimental short - term effects . putting aside the difficulties of integrating an instantaneously acquired innovation into a cellular network , it is universally understood that some types of laterally transferred genes can have enormous positive fitness impacts on a bacterial cell . an obvious example is antibiotic resistance . with the clinical introduction of antibiotics in the mid 20th century the rapid emergence and spread of antibiotic resistance was one of the earliest indicators of evolution by lgt . today , the ubiquitous presence of multi drug resistant bacteria has led the world health organization to recognize the resulting decline in antibiotic efficacy as one of the great health challenges of the 21st century . most identified antibiotic resistance genes are part of the mobile genome and the selection for antibiotic resistance has facilitated the assembly and concentration of the genes conferring resistance into a plethora of mobilizing elements . this enormous resistance gene diaspora can be attributed to strong artificial selection by human activities and is very recent . the presence of these laterally transferred genes in diverse bacteria is relatively easy to understand . thus , in many contexts , the fitness of cells lacking resistance genes is essentially zero . also , resistant bacteria are predicted to persist in the environment even if all uses of antibiotics ceased . the reasons for persistence in the absence of selection are not all entirely clear although one explanation for the reduced fitness cost is that most acquired resistance genes or gene pathways act autonomously and outside cell metabolic networks . this in itself helps to account for the rapid dissemination of genes through diverse pathogens since a resistance gene will work in any cellular context . thus , the same gene can be found to be mediating resistance in many disparate pathogens . prior to the genomic era , the bulk of our understanding of lgt was drawn from highly mobile genes and the elements that mobilized them . lgt was then considered a marginal process which did not concern most genes , including those used to reconstruct a phylogenetic universal tree of life . the compatibility of our understanding of prokaryotic evolution to neo - darwinian concepts was not questioned , as lgt was only viewed to affect a minority of specialized genes . the microbial genomics era , which began in 1995 , with every passing year and additional sequenced genome , progressively led to the incontrovertible conclusion that lgt has been a dominant force in microbial evolution . thus , rather than just adding on genes in response to strong selection that act independent of the cell network , laterally transferred genes were key integrated components in the cell . several recent studies have shown that phylogenies of gene families that are involved in cell networks do not conform to a simple bifurcating tree type model as would be expected for vertical inheritance . pal et al . have argued that lgt has played an important role in building up complex networks via involvement in pathways that allow bacteria to niche adapt to specialized environment types . thus , an influx of genes at the periphery is most likely to assist bacteria in adapting to new environments and not by optimization in fixed environments . this model is attractive in that it is consistent with the diverse ecological niches that prokaryotes have come to inhabit . by analogy with the complexity hypothesis , this model predicts that metabolic genes rather than core informational genes are more likely to show evidence of lgt . there is clear evidence that this is true in a relative sense , however it is also the case that some informational genes also display evidence of frequent lgt . even if mostly added at the periphery , it is clear that newly acquired genes need to be able to communicate with existing cell networks to some extent even if only , for example , to channel partially processed substrates into central metabolic networks . it has thus been argued that the evolution of new integrated pathways requires the evolution or acquisition of regulatory proteins as well as enzymatic ones . understanding how networks are impacted by lgt is crucial to fully integrating this phenomenon in evolutionary theory . it also has relevance to more applied branches of science , as cellular networks are critical in the evolution of pathogenicity and impact on the evolution of pathogenic bacteria . thus , although the genomics era has told us a lot about the extent of lgt and where it has occurred , as pointed out by davids and zhang , plausible mechanisms as to the events that lead to integration of acquired genes are lacking . for several years our laboratories have been investigating the biology of the integron / gene cassette system . this system is an important component of the mobile genome in gram - negative bacteria . it was first characterized in the context of its role in spreading antibiotic resistance genes in human pathogens . in that regard , it is an exemplar of the power of the adaptive potential of mobile dna since , although it was selected for and spread as a result of the heavy use of antibiotics by humans , the integron is an ancient structure that has been a feature of many bacterial genomes for a long period of evolutionary time . the defining feature of all integrons is their ability to capture genes when the latter are part of mobilizable elements known as gene cassettes . what makes integrons arguably unique in the plethora of mobile elements is that they appear to be highly adapted as a tool kit for natural experimental evolution . their key feature is their ability to insert any gene cassette at a defined integron associated recombination site by site - specific recombination . the advantage of this process is that it allows insertion of a dna sequence at a defined location in the genome which otherwise does not disrupt any other gene in the cell by insertional inactivation . it also allows the immediate expression of the newly acquired gene , as a promoter is located next to the insertion site . unlike other site specific recombination systems however , insertion does not involve a single discrete dna sequence such as a lysogenic phage but rather , a diversity of sequences most of which include defined genes , that to date has no definable upper limit in terms of numbers . thus mobilized genes acquired by lgt can be inserted and expressed in a way that does not otherwise impact on cellular gene content . other evidence that this system is designed to facilitate adaptive innovation is the fact that the site - specific recombination reaction is genetically regulated such that the sos response leads to an increase in mobile cassette rearrangement frequencies . therefore , this provides a mechanism for generating diversity at times when cells have to rapidly adjust to new or changing environments . apart from the large number of mobile genes known to exist , another defining feature of the cassette metagenome is the extraordinary amount of novelty it contains . this novelty extends to the point that most genes found within the cassette metagenome either possess no identified homologs or are homologous to genes encoding proteins that are identified only as one of the great challenges in this area of research is in understanding what these mobile genes do , especially given the fact that they , collectively , comprise a resource that must be many orders of magnitude larger than any single bacterial genome . in our view , however , there is no question that the vast majority of cassette - encoded proteins are adaptive . for example , structural biology approaches ( by obtaining high resolution crystal structures ) have revealed functions for many such proteins . thus , we have found via these approaches that some cassette proteins include putative house cleaning functions and ligand binding domains commonly associated with two component transcriptional regulators both of which are likely to impact on cell networks . this latter example is particularly interesting as it implies that the modular rearrangement of protein domains via cassette shuffling may be a precursor for the evolution of multi domain proteins . one could envisage a scenario whereby two adjacent cassettes providing complementary functions may become fused by loss of the cassette recombination site creating a new multi domain protein . cassette fusion has been previously observed . in any event , the notion that mobile cassettes may encode transcription regulators as well as enzymatic proteins is consistent with this genetic element constituting an adaptive toolbox . apart from potentially evolving new proteins , cassette uptake in natural environments and shuffling may be a process for operon creation by bringing together functionally distinct proteins that can cooperate to form a co - regulated biochemical pathway . other approaches besides structural biology have been used to understand what role cassette - encoded proteins play in the cell . our major model for this is the vibrio rotiferanus strain dat722 , the genome of which we have recently sequenced . from an integron perspective , this species is typical of the vibrios in that it has large cassette arrays composed of 116 cassettes in the case of dat722with most of the associated proteins having no identifiable function . this makes it and its close relatives useful models for testing of specific hypotheses . in a recent study we examined the impact of cassette array deletions on the metabolic capacity of the dat722 cells this was initially done by examining the ability of the mutants to grow on a variety of carbon sources in a biolog screening assay . our intent was to try and identify mutants with a varied capacity to metabolize specific substrates . surprisingly , we found that some mutants concomitantly had a greatly reduced viability in minimal media in the presence of a number of different carbon substrates including glucose . furthermore , these mutants exhibited a hypermutative phenotype ( labbate m. , unpublished ) indicating the deletion had resulted in the loss of a significant gene which made the bacterium maladapted to its environment . this change of phenotype was ascribed to one specific protein , encoded within cassette 11 ( the 11th cassette in the 116 cassette array ) . the cassette 11 protein was demonstrated to have a role in porin regulation and the altered growth profiles were a consequence of changes in porins . the reduction in fitness on deletion of cassette 11 is substantial to the point of making the cell nearly non viable in certain carbon - containing minimal growth media . to our knowledge this is the first experimental data that demonstrates the integration of an apparently unique mobile gene into an important cell network . one particularly interesting aspect to this is the fact that the negative impact on growth is specific for a media that closely resembles the environment in which free living vibrios are most commonly found namely estuarine water . to test whether cassette 11 protein can impact on the fitness of other vibrio strains , we introduced the cassette 11 gene containing recombinant vector pmaq1082 into a v. cholerae strain designated s25 to determine whether fitness was affected . pmaq1082 comprises the cloning vector pjak16 into which the cassette 11 gene has been cloned under the control of an iptg inducible promoter . s25 is an environmental nono1/nono139 v. cholerae strain isolated from a sydney , australia , estuarine environment . it has a large integron array but does not possess cassette 11 or a close homolog based on pcr using primers targeting this cassette . 1 ) in complete media a result identical to that for dat722 with and without this cassette . in contrast to dat722 however , which had greatly altered growth rates in most minimal media , including 2 m + glucose , when isogenic strains with and without cassette 11 were compared , the growth of s25 in the same media was unaffected by the presence or absence of this cassette and was identical to the growth seen in complete media ( fig . 1 ) . the simplest interpretation of this is that the cassette 11 protein does not interact with any s25 cell networks in stark contrast to its influence on networks in dat722 . while more data are needed , we speculate that integration of some mobile genes into cell networks may be analogous to the evolution of duplicated genes in eukaryotes . in eukaryotes duplication is most commonly via the generation of identical copies of an existing gene . in contrast , the introduction of cassette 11 into the ancestor of dat722 initially may have had no impact on the cell as this progenitor possessed a non identical gene that nonetheless encoded a protein with a related function . as this strain evolved however incremental changes led to cassette 11 protein replacing this pre existing protein in terms of its central network role . v. cholerae s25 ( pmaq1082 ) may be a useful experimental evolution model for exploring this hypothesis . growth curves of v. cholerae s25 ( squares ) , v. cholerae s25/pjak16 ( circles ) and v. cholerae s25/pmaq1082 ( triangles ) induced with 0.1 mm iptg in lb20 ( a ) and 2 m + glucose ( b ) . what is the cassette 11 encoded protein ? at this time we do not have a definitive answer although its main target is most likely dna and consequently it may play a role in regulation of dna supercoiling . this potential link is inferred by the presence of two distinct domains in the protein . one of these is a c - terminal zinc finger domain commonly associated with prokaryotic dna topoisomerase i proteins and in these proteins catalyzes the relaxation of supercoiled dna . the second is a recently identified nuclease related nerd domain inferred to have a role in dna processing . we have found this domain is present in proteins found in diverse bacteria ( fig . 2 ) , a distribution suggestive of spread by lgt as is the case for the cassette 11 protein family overall . interestingly this bioinformatic analysis reveals examples of this nerd domain being encoded by genes in highly mobilized elements ( gene cassettes and transposons ) as well as being fixed in cell lines for ( presumably ) longer periods of time as a result of the gene being located on a chromosome . obtaining a crystal structure and identifying a precise biochemical function of the cassette 11 protein would be of great interest . an implied role in dna processing is tantalizing as this type of information processing function is one of the least likely candidates for successful lgt according to the complexity hypothesis or the network evolution model advanced by pal . understanding its precise role is likely to shed light on the forces that allow and provide for rapid integration of lgt derived genes into cell networks . if a gene can be integrated into a cell line specific network such that its loss is nearly fatal , this integration event may represent the first step in a process that represents sympatric speciation . each taxon name is followed by the accession number of the protein it contains , as well as the genetic element the protein is associated with . confidence values over 80% are displayed on nodes of interest , representing the proportion of bootstrap pseudo - replicates supporting topology . in summary , core prokaryotic genes defined by little identifiable evidence of lgt over long evolutionary periods constitute only a small minority of the genes in extant genomes . the majority of genes have at some point been acquired by lgt , including those that are now fixed on chromosomes and integrated into the cell networks that support prokaryotic life . it is these genes that have allowed niche specialization and adaptation of bacteria to novel environments . a complete understanding of the process of lgt requires an understanding of how new genes integrate into cell networks . this systems biology understanding will require hypothesis driven experimental approaches as well as those involving genomics and bioinformatics .

despite the prophylaxis and preemptive strategies using potent antiviral agents , cytomegalovirus ( cmv ) remains a major infectious cause of morbidity and mortality in allogeneic stem cell transplantation ( sct ) recipients . delayed immune reconstitution after sct , such as cord blood and t - cell depleted sct with the use of alemtuzumab , has been associated with an increased frequency of cmv disease as well as cmv reactivation . cmv disease involving central nervous system is an unusual presentation in the setting of sct . we report a case of cmv ventriculoencephalitis after unrelated double cord blood sct with an alemtuzumab - containing preparative regimen for philadelphia - positive acute lymphoblastic leukemia . lateral gene transfer ( lgt ) impacts on the evolution of prokaryotes in both the short and long - term . the short - term impacts of mobilized genes are a concern to humans since lgt explains the global rise of multi drug resistant pathogens seen in the past 70 years . however , lgt has been a feature of prokaryotes from the earliest days of their existence and the concept of a bifurcating tree of life is not entirely applicable to prokaryotes since most genes in extant prokaryotic genomes have probably been acquired from other lineages . successful transfer and maintenance of a gene in a new host is understandable if it acts independently of cell networks and confers an advantage . antibiotic resistance provides an example of this whereby a gene can be advantageous in virtually any cell across broad species backgrounds . in a longer evolutionary context however laterally transferred genes can be assimilated into even essential cell networks . how this happens is not well understood and we discuss recent work that identifies a mobile gene , unique to a cell lineage , which is detrimental to the cell when lost . we also present some additional data and believe our emerging model will be helpful in understanding how mobile genes integrate into cell networks .

diabetes mellitus ( dm ) is a clinical syndrome characterized by hyperglycemia because of absolute or relative deficiency of insulin . the diagnosis of dm is based on blood glucose estimations . blood collection is an invasive procedure , and may be traumatizing , especially in diabetic patients who require routine daily monitoring of blood glucose levels . ongoing research in the past few decades has focussed on alternative methodologies that involve incorporating various other body fluids that could be used as a substitute for blood for diagnostic purposes . alterations in the salivary flow and composition of saliva in diabetics have been reported in numerous previous studies , although the findings have frequently been contradictory . there still is no consensus about which parameters should be followed in saliva of type 2 dm patients to enable a salivary diagnosis of type 2 dm . participants were informed about the study protocol , and only those who provided their written consent were included in the study . this cross - sectional study was conducted over a period of 8 months from june 2010 to february 2011 . based on the available literature of cross - sectional , observational studies which included sample sizes that ranged 40180 , in this study the sample size was considered to be 100 , which included 60 diabetics and 40 healthy controls . sixty patients previously diagnosed with type 2 dm and with no other systemic illness , and 40 healthy volunteers with no apparent medical history in the age group of 3060 years were randomly selected and included in the study . completely edentulous patientspatients with any oral mucosal lesionspatients on any medications other than for type 2 dmtobacco / betel chewing habits . completely edentulous patients patients with any oral mucosal lesions patients on any medications other than for type 2 dm tobacco / betel chewing habits . random capillary blood glucose ( rcbg ) was estimated using sterile lancets and sd check gold glucometer with glucose reagent strips using the finger prick method . saliva samples were collected in the morning between 9 am and 12 pm . before collecting the saliva samples , they were asked to spit into the graduated disposable collecting cup at the end of every minute for 5 minutes and the average was calculated to estimate the unstimulated whole salivary flow rate . salivary ph was estimated by placing the gc saliva ph strip for 10 seconds in the saliva sample , which was then removed and matched with the color - coded table provided along with the kit . pipettes provided along with the kit were used to draw saliva sample from the collecting cup and 3 drops added over the 3 slots of the strips . after 2 minutes , the color change was matched with the color - coded table scores provided by the manufacturer . decayed missing filled teeth ( dmft ) index was recorded to assess the status of teeth . using a micropipette , 10 l of saliva was drawn from the disposable collecting cup and added into a cuvette to which 1000 l of glucose oxidase peroxidase enzyme reagent was added ; the sample was then incubated at 37c for 10 minutes . similarly , 10 l of standard glucose solution was drawn into a cuvette to which 1000 l of enzyme reagent was added , and the sample was incubated at 37c for 10 minutes . the optical absorbance readings were recorded using the digital photocolorimeter using the green filter with a peak of 540 nm wavelength [ figure 1 ] . armamentarium used to assess unstimulated whole salivary glucose uwsg was calculated using the formula : salivary glucose in mg / dl = absorbance of sample concentration of standard / absorbance of standard concentration of the standard glucose was 100 mg / dl statistical analysis was done using contingency coefficient analysis , independent samples t - test , multivariate analysis of variance ( manova ) , and correlations using pearson coefficient . the statistical package for the social sciences ( spss ) for windows ( spss , version 16.0 , chicago , spss inc . ) was used for statistical analysis . sixty patients previously diagnosed with type 2 dm and with no other systemic illness , and 40 healthy volunteers with no apparent medical history in the age group of 3060 years were randomly selected and included in the study . completely edentulous patientspatients with any oral mucosal lesionspatients on any medications other than for type 2 dmtobacco / betel chewing habits . completely edentulous patients patients with any oral mucosal lesions patients on any medications other than for type 2 dm tobacco / betel chewing habits . random capillary blood glucose ( rcbg ) was estimated using sterile lancets and sd check gold glucometer with glucose reagent strips using the finger prick method . saliva samples were collected in the morning between 9 am and 12 pm . before collecting the saliva samples , patients were asked to rinse their mouth with 200 ml water . they were asked to spit into the graduated disposable collecting cup at the end of every minute for 5 minutes and the average was calculated to estimate the unstimulated whole salivary flow rate . salivary ph was estimated by placing the gc saliva ph strip for 10 seconds in the saliva sample , which was then removed and matched with the color - coded table provided along with the kit . pipettes provided along with the kit were used to draw saliva sample from the collecting cup and 3 drops added over the 3 slots of the strips . after 2 minutes , the color change was matched with the color - coded table scores provided by the manufacturer . decayed missing filled teeth ( dmft ) index was recorded to assess the status of teeth . using a micropipette , 10 l of saliva was drawn from the disposable collecting cup and added into a cuvette to which 1000 l of glucose oxidase peroxidase enzyme reagent was added ; the sample was then incubated at 37c for 10 minutes . similarly , 10 l of standard glucose solution was drawn into a cuvette to which 1000 l of enzyme reagent was added , and the sample was incubated at 37c for 10 minutes . the optical absorbance readings were recorded using the digital photocolorimeter using the green filter with a peak of 540 nm wavelength [ figure 1 ] . armamentarium used to assess unstimulated whole salivary glucose uwsg was calculated using the formula : salivary glucose in mg / dl = absorbance of sample concentration of standard / absorbance of standard concentration of the standard glucose was 100 mg / dl statistical analysis was done using contingency coefficient analysis , independent samples t - test , multivariate analysis of variance ( manova ) , and correlations using pearson coefficient . the statistical package for the social sciences ( spss ) for windows ( spss , version 16.0 , chicago , spss inc . ) was used for statistical analysis . the mean rcbg and uwsg levels in type 2 diabetics were 180 mg / dl and 12.9 mg / dl , respectively [ table 1 ] . the mean rcbg and uwsg levels in healthy controls were 95.1 mg / dl and 9.46 mg / dl , respectively [ table 1 ] . the difference between the groups was statistically significant ( p = 0.000 ) [ table 2 ] . descriptive statistics independent samples test : control and type 2 dm a positive correlation between rcbg and uwsg was observed in both the study and control groups [ graphs 1 and 2 ] . the mean unstimulated whole salivary flow rate in type 2 diabetics was 0.6 ml / min and in the healthy controls it was 0.67 ml / min [ table 1 ] . the difference in unstimulated whole salivary flow rate between the groups was statistically significant ( p = 0.029 ) [ table 2 ] . the mean unstimulated salivary ph in type 2 diabetics was 6.8 and in the healthy controls it was 7.1 [ table 1 ] . scatter plot no.1 correlation between random capillary blood glucose and unstimulated whole salivary glucose in control group scatter plot no 2 : correlation between random capillary blood glucose and unstimulated whole salivary glucose in experimental group the mean salivary buffering capacity in type 2 diabetics was 7 and in the healthy controls it was 8.4 [ table 1 ] . there was a statistically significant difference in the salivary buffering capacity between the groups ( p = 0.021 ) [ table 2 ] . no significant correlation between salivary flow rate and the salivary buffering capacity was observed in type 2 diabetics , however , a statistically significant correlation ( p = 0.000 ) was found between salivary flow rate and salivary ph [ table 3 ] . correlations in the study group ( type 2 diabetics and healthy volunteers ) among various parameters the values of salivary ph and salivary buffering capacity showed good correlation in both the type 2 diabetics and the control group , as the salivary ph decreased the salivary buffering capacity also decreased and the relationship was highly significant statistically ( p = 0.000 ) [ table 2 ] . the mean values for rpi in the type 2 diabetics was found to be 2.1 , and in the healthy controls it was 1.2 [ table 1 ] . the difference in the rpi scores between the groups was significant statistically ( p = 0.000 ) [ table 2 ] . the mean dmft scores in type 2 diabetics was 5.7 and in the healthy controls it was 5.8 [ table 1 ] . the rpi scores of type 2 diabetics showed positive correlation with only the salivary glucose levels among the various tested parameters . none of the tested salivary factors showed any statistically significant effect on the rpi scores in the control group . the caries experience of both type 2 diabetics and controls in our study was similar with no statistical difference in the dmft scores [ table 3 ] . epidemiological studies in india have shown high prevalence of type 2 dm ; in the year 2002 , it was estimated that there were 19.4 million individuals affected by type 2 dm , which is likely to increase up to 57.2 million by the year 2025 . routine blood examination for glucose assessment can be traumatizing to the patient , and hence , other alternatives have been explored , among which salivary diagnostics hold much promise . saliva - based diagnostics are not limited to oral diseases but have been extended to the entire physiologic system , as most compounds found in the blood are also present in the saliva . accordingly , saliva can reflect the physiologic state of the body including emotional , endocrinal , nutritional , and metabolic variations , and acts as a source for monitoring oral and systemic health . a systematic review of previously published studies reflects the fact that salivary glucose concentration increases in type 2 dm , and a positive correlation exists between blood glucose and salivary glucose ; hence , it can be a useful biomarker to monitor type 2 dm . in the present study type 2 diabetics had significantly higher uswg / rcbg levels than the controls , a fact which has been documented in previous studies . the correlation between rcbg and uwsg could plausibly be because of leakage of glucose from blood across the basement membrane of salivary glands . microvascular alterations in the blood vessels that are commonly seen in type 2 diabetics could also contribute to increased salivary glucose levels . saliva samples collected in the present study represented the whole mouth fluid , and therefore , reflects glucose levels not only due to leakage across the basement membrane of major and minor salivary glands but also from the gingival crevicular fluid . furthermore , it has been proposed by belazi that the basement membrane alterations lead to enhanced leakage of serum components including glucose into the gingival crevicular fluid rather than into saliva . however , in contrast to the present study , various other authors could not establish any correlation between rcbg and uwsg . the decrease in the unstimulated whole salivary flow rate in type 2 diabetics is in accordance with previous studies . type 2 dm is known to affect the sympathetic and parasympathetic nervous system of the salivary glands , resulting in decreased salivary secretion , microangiopathy , dehydration , and hormonal changes , which may contribute to the decrease in the salivary flow rate . however , few authors were not able to establish significant difference in salivary flow rates between type 2 dm and healthy controls . we found a significant difference in the salivary ph between the type 2 dm patients and control ( p < 0.01 ) , which was similar to other studies . in accordance with previous studies , we found significant differences ( p < 0.05 ) in the buffering capacity between type 2 dm and control groups . this can also be attributed to the hormonal and metabolic changes in diabetic patients causing altered levels of salivary buffering systems . results contrary to our study have been reported by collin et al . in the present study , there was significant correlation ( p < 0.01 ) between the salivary flow rate and salivary ph in the diabetics , and such correlation was not observed in the control group individuals . even though type 2 dm patients had significant decrease in salivary flow rate in our study , it was observed that salivary flow rates were not as low as those in patients suffering from hyposalivation . this could be caused by increased fluid intake by diabetics due to polydipsia . because buffering capacity is dependent on the ph levels , type 2 dm had salivary buffering capacity correlating with the salivary ph . interestingly , it was observed that , in the control group , salivary ph levels were within the normal limits independent of the salivary flow rate . this could be due to reduced acidogenic flora in the oral cavity and increased salivary clearance activity maintaining normal ph levels . reported that , in patients with type 2 dm , the risk of periodontal disease is three times higher than that in the general population . similarly , we found that the type 2 dm patients had significantly poor periodontal status than the healthy controls . it has been shown that dm causes alterations in the connective tissue metabolism by uncoupling the resorptive and formative processes , thus leading to increased levels of loss of periodontal attachment and bone loss . among all the parameters tested in our study , only rcbg and uwsg showed significant positive correlation ( p < 0.01 ) with the rpi scores in type 2 dm patients . none of the other salivary parameters studied correlated with the rpi scores , indicating that the level of glycemic control is an important determinant in being a risk factor for the development of gingivitis and periodontitis in type 2 dm . studies concerning the occurrence of caries in diabetic patients have yielded controversial results . in the present study , no significant difference was observed in the dmft scores between the type 2 dm and controls , similar to earlier studies . lack of significant difference in the dmft scores between the groups could be due to modification in the diet with reduced amounts of refined carbohydrate intake by the type 2 dm patients , thereby reducing the formation of an acidogenic environment . the fact that most of the patients who formed the study group belonged to the urban population and had unproblematic access to dental care could have also contributed to no significant differences in the mean dmft scores between the type 2 dm and control groups . our results are contrary to a few authors who have reported that diabetics have slightly higher mean dmft scores than the controls . from our results , it can be concluded that the salivary glucose levels reflect the random blood glucose levels . type 2 diabetics have significantly lower salivary flow rate , ph , and buffering capacity and present with advanced periodontal destruction than the healthy population . further studies with larger sample size are warranted to substantiate the correlation between blood glucose and salivary glucose to devise saliva - based tests for diagnosing dm . due to increasing life expectancy of the dentition , older adults are experiencing root caries and gingival recession , putting them at even higher risk for periodontal disease . root caries is the major cause of tooth loss in older adults , and tooth loss is the most significant oral health - related negative variable of quality of life for the elderly . one prominent goal of the dental profession is to preserve and maintain dentitions throughout life . population projections suggest that the proportion of the population aged 65 years and older will nearly double between 2000 ( 12.6 percent ) and 2030 ( 20.0 percent ) , and that the proportion of those aged 85 years and older will increase dramatically over the next 10 to 15 years . this population trend coupled with compelling evidence that people are retaining their teeth into old age suggests that there will be an increased number of older adults with many more natural teeth in the years to come . there are known clinical and behavioral risk factors involved in the production and progression of root caries in the elderly . risks are described in a number of levels , from socioeconomic status to salivary flow to presence of dentures . data have shown correlations of dietary and oral habits and other variables on root caries . many risk factors can compromise an older adult 's systemic health such as sociodemographic variables , nutrition / diet , and weakened immune system . this paper examines salivary hypofunction , the systemic and oral immune system ( immunoglobulins found in saliva ) in older adults , and their manifestations . there are several indicators that provide insight into the incidence and prevalence of caries in healthy people and the medical or disability conditions that place individuals at increased caries risk . one indicator is the presence of mutans streptococci , an established etiologic agent for caries activity . one of the main oral behaviors to reduce the amount of bacteria in the oral cavity is regular tooth brushing with a fluoride - containing dentifrice . conditions that compromise good oral hygiene behaviors and oral health are also positively associated with caries risk . these include certain illnesses , physical and mental disabilities , and the presence of existing restorations or oral appliances . fermentable carbohydrate consumption fuels acid formation and demineralization and is associated with caries , particularly in the absence of fluoride . long - term regular doses of medications containing glucose , fructose , or sucrose may also contribute to caries risk . medical conditions such as sjgren 's syndrome , pharmacological agents with xerostomic side effects , and therapeutic radiation to the head and neck lower salivary flow rate to pathological levels and dramatically elevate a patient 's risk of caries . some studies indicate that low buffering capacity , low salivary immunoglobulin a , and low salivary calcium and phosphate may also be linked to increased caries . the inability to maintain good oral hygiene and xerostomia are risk factors of special significance among the elderly , and gingival recession uniquely increases the risk of root caries in elderly populations by exposing previously protected root surfaces to cariogenesis . low indices of socioeconomic status have been associated with elevation in caries and are also associated with reduced access to care , reduced oral health aspirations , low self - efficacy , and health behaviors that may enhance caries risk . older age is positively associated with the prevalence of root caries . over half evidence also suggests that adults who have lived in fluoridated areas throughout most of their lives , including the time of tooth formation , have a lower prevalence of root caries . there appear to be a wide variety of risk indicators and risk factors implicated in root caries . these factors include not only oral factors , but also medical , behavioral , and social factors . it is likely that numerous microbial , genetic , immunological , behavioral , and environmental contributors to risk are at play in determining the occurrence and severity of clinical disease . prevention and treatment can be achieved by identifying and arresting or reversing the disease at an early stage . treatments include application of fluorides , chlorhexidine , sealants , antimicrobials , salivary enhancers , and patient education . studies have shown that one 's preference to sweet carbohydrates may put one at risk for caries . this preference may be determined by socioeconomic status but also be under genetic control . studies have examined genetically determined taste sensitivity to 6-n - propylthiouracil demonstrating that individuals with low taste sensitivity experience a lower caries risk than those with high tasting sensitivity [ 16 , 17 ] . the examination of genetic variation in taste pathway genes ( taste receptor , type 2 , member 38 ( tas2r38 ) , taste receptor type 1 member 2 ( tas1r2 ) , and guanine nucleotide - binding protein g(t ) subunit alpha-3 ( gnat3 ) ) and their relation to caries revealed some associations . tas1r2 is a member of sweet taste receptor family , and gnat3 codes for the g protein gustducin , which mediates taste receptor signaling in the taste buds of the lingual epithelium . a significant association was found for certain alleles in tas2r38 that were protective from caries , while other haplotypes were associated with caries risk . this association held true only for the primary dentition with individuals with a mean age of 3.4 years . there was no significant association in the mixed and permanent dentitions , which had individuals with mean ages of 9.8 and 29.4 years , respectively . the tas2r38 single nucleotide polymorphisms that were found to be protective for caries cause amino acid changes in the taste receptor that are associated with bitter sensitivity . variations of the genetic makeup of these genes may contribute to differences in dietary habits that influence the caries risk of these children . evaluation of children classified with different tasting abilities has also been associated with body weight and dietary habit differences . at this point , these studies have not targeted the elderly and/or root caries . to support the notion that caries is a disease with a genetic component , one study used dna samples collected from 110 individuals older than 12 years of age from guatemala and documented who had a higher or lower caries experience using dmft ( decayed , missing due to caries , filled teeth ) scores . enamel proteins such as ameloblastin and tuftelin are associated and crucial for proper enamel formation . single - nucleotide polymorphism markers were genotyped in selected candidate genes ( ameloblastin , amelogenin , enamelin , tuftelin-1 , and tuftelin interacting protein 11 ) that influence enamel formation . having at least one copy of the rare amelogenin marker allele was associated with increased age - adjusted caries experience . this association was stronger in individuals with higher dmft ( dmft 20 ; p = .0000001 ) , suggesting that a variation in amelogenin may contribute to caries susceptibility in the population studied . these results were confirmed in an independent cohort from turkey . besides genes related to taste preferences and enamel formation , three single nucleotide polymorphisms in defb1 ( beta defensin 1 ) were tested in a cohort of unrelated adult individuals . carrying a copy of the variant allele of the defb1 marker rs11362 increased the dmft and dmfs scores more than fivefold . also , carrying a copy of the variant allele of the defb1 marker rs179946 correlated with low dmft scores . a high caries experience promoter haplotype ( gca ) increased dmft scores twofold , and a low caries experience promoter haplotype ( acg ) decreased dmft scores two - fold . as we age our immune system weakens and fewer antimicrobial immunoglobulins are produced and found in saliva . medications that are prescribed to the elderly in fact can cause impaired salivary flow with no change in the immune system . many medications , chemotherapy , radiation treatments , and some diseases can decrease salivary gland function and therefore make caries and other oral diseases more likely to occur . some common drugs that may cause dry mouth are high blood pressure drugs , cholesterol lowering drugs , pain medications , muscle relaxants , allergy , and asthma medications . no matter what the cause , it is undisputed that saliva is essential in neutralizing the acidic environment , thus inhibiting the growth of bacteria . any decreased levels of saliva can put one at increased risk for developing caries . when studying the elderly population , researchers have looked at age - related differences in whole and parotid saliva secretion related to the production of saliva , as well as the immune factors in saliva . the levels of serum immunoglobulin g ( igg ) and igm were significantly reduced in older individuals , whereas no significant reduction in the level of iga with age was observed . no significant changes in any immunoglobulin levels with age were found in parotid saliva , but significant reductions in the secretion rates of iga and igm , but not igg , in whole saliva were detected in the oldest age individuals . the results demonstrate a decline in immunoglobulin concentrations with increased age , which may contribute to the increased susceptibility of elderly individuals to oral diseases . one hundred and sixty of the 1,328 parotid gland genes show more than a twofold change in expression . the effects of age on specific gene expression in the human parotid gland may provide insight into functional and morphological changes in the oral cavity and its associations to oral disease . when examining unstimulated and stimulated submandibular / sublingual saliva flow rates , unstimulated and stimulated parotid saliva flow rates , and different proteins ( lactoferrin , secretory iga , albumin , lysozyme , mucin , and cystatin ) , significant associations were found between caries , age , and specific individual submandibular / sublingual salivary protein levels . therefore , these changes in saliva components over age may represent caries risk indicators . age has a significant influence on the expression of genes associated with reduced protein biosynthesis of salivary gland secretion . the expressions of both hla - dqa1 ( major histocompatibility complex , class ii , dq alpha 1 ) and hla - dqb1 ( major histocompatibility complex , class ii , dq beta 1 ) , genes involved in immuneresponse , were decreased in the parotid gland in the elderly . several other proteins known to be involved in different immune response pathways showed altered expression in aged population ( e.g. , irf1 , irf7 , gbp1 , ifitm1 , ifitm2 , ifitm3 , psmb8 , and psmb9 ) . complex remodeling of the immune system occurs during aging , which may contribute significantly to systemic diseases in the elderly . diseases such as infections , autoimmune , and neoplastic pathologies that aged individuals are particularly susceptible to involve dysregulation of immune function . the number of elderly is dramatically increasing , and consequently , geriatric pathology is becoming a more important aspect of clinical practice . in light of this , salivary gland function may prove to be a risk factor worth evaluating in the elderly . diet is a very important factor in preventing caries since certain foods and snacks can greatly increase the number of bacteria that forms the decay - causing plaque . the more sweetened snacks consumed and the more frequently they are consumed increase the risk for developing caries . the frequency of sugar intake is more important than the amount of sugar consumed in the development of caries . therefore , minimizing snacking is recommended since snacking creates a continual supply of nutrition for acid - creating bacteria in the mouth . also , chewy and sticky foods ( such as dried fruit or candy ) tend to adhere to teeth longer and consequently are best eaten as part of a meal . when studying the elderly population , it is beneficial to look at other factors such as diet , which together with decreased salivary flow make one more susceptible to root caries . when dietary habits , microbial factors , and salivary factors were analyzed together in older adults who had root caries compared to adults who did not have root caries , individuals with root caries ate a greater number of meals a day and had higher sugar intake . root caries subjects had significantly higher lactobacilli counts and less salivary buffering capacity suggesting that higher microbial counts and less salivary flow may be risk factors associated with root caries in older adults . plaque consists of bacteria and an extracellular matrix that contains lipids , proteins , and polysaccharides . teeth are more vulnerable to an increase in bacterial plaque when carbohydrates in the food are left on teeth after every meal . in the presence of sugar and other carbohydrates , bacteria in the mouth produce acids that can demineralize enamel , dentin , and cementum . the more frequently teeth are exposed to this environment , the more likely caries are to occur . certain bacterial species appear to be strongly associated with health , as they are rarely detected or are absent from root caries carriers but are commonly found in healthy subjects . in root caries , veillonella parvula , veillonella dispar , selenomolas noxia , campylobacter gracilis , streptococcus mutans , selenomonass putigena , and fusobacterium nucleatum are found at high levels . lactobacilli appears to be associated with disease , as they are common in carious lesions , while rare or absent in healthy teeth . in individuals with no caries , streptococcus mutans are less common and lactobacilli are absent , while for individuals with root caries , levels of streptococcus mutans and lactobacilli are increased . the prevalence of streptococcus mutans alone or in combination with lactobacilli is similar in root caries lesions . lactobacilli are absent in healthy subjects but highly present in carious dentin , supporting the suggestion that lactobacilli might play a significant role in the progression of root caries . bacterial species typically associated with root caries can be detected , such as streptococcus mutans , lactobacilli , and actinomyces . although many variables influence the production and progression of oral disease , the one variable that shows an immediate and long lasting significant effect on one 's oral health is oral hygiene . the purpose of oral hygiene ( brushing and flossing daily ) is to minimize , remove , and prevent the formation of plaque . three monthly applications of chlorhexidine - thymol varnish ( cervitec ) over one - year limits the progress of existing root caries lesions and reduces the incidence of root caries . when fluoride varnish , 1% chlorhexidine , 40% chlorhexidine , and professional tooth cleanings were compared in regard to root caries , all methods showed significant reduction in the amount of microbiota ( bacteria ) . these data suggested that tooth cleaning alone might be as effective in reducing plaque formation ( and subsequently root caries ) as fluoride or chlorhexidine . one of the more groundbreaking studies of oral disease today is examining the associations between oral and systemic diseases . data from the national health and nutrition examination survey 19992004 showed that individuals with rheumatoid arthritis , diabetes , or a liver condition were twice as likely to have an urgent need for dental treatment . the data also showed that arthritis , cardiovascular diseases , diabetes , emphysema , hepatitis c , obesity , and stroke were all associated with dental disease . others may interpret this association as meaning that those with systemic disease tend to neglect their oral health and so show a higher incidence of oral disease . in an attempt to evaluate whether self - reported systemic diseases were associated with caries experience , data from the university of pittsburgh school of dental medicine dental registry and dna repository regarding medical history and caries experience ( dmft and dmfs ; decayed , missing due to caries , filled teeth / surface ) were analyzed . an association was found between higher caries experience ( dmft above 15 and dmfs above 50 ) and asthma and epilepsy . cardiovascular diseases have also been associated with higher caries experience , particularly in individuals 80 years or older . individuals with three or more active root caries lesions have more than twice the odds of cardiac arrhythmias than ones without active root caries . these results did not notably change after adjusting for age , medications that reduce saliva , and number of teeth . the findings indicate that there may be a link between active root caries and cardiac arrhythmias in those aged 80 and older . one explanation for these findings is that both cardiac arrhythmias and caries are simply markers of declining general health . xerostomia , commonly associated with oral disease , has also been associated with type 2 diabetes mellitus . the prevalence of xerostomia is higher ( 62% ) in subjects with type 2 diabetes mellitus in comparison to the nondiabetic controls ( 36% prevalence ; p = .001 ) . in the same way , the prevalence of hyposalivation is higher in individuals with type 2 diabetes mellitus ( 46% ) , whereas only 28% of the controls had hyposalivation ( p = .03 ) . subjects with hyposalivation had significantly higher numbers of mutans streptococci , lactobacillus , and candida in the saliva compared to those without hyposalivation . the higher number of pathogens and decreased salivary flow may very well explain why diabetics have or are at higher risk for oral disease . these papers revisit aspects related to diet , microbial colonization , oral hygiene , and concomitant systemic illnesses , as well as several topics not covered in this section , such as nonimmunoglobulin salivary agents , chewing ability , sugar clearance , antimicrobial mouthwashes , saliva substitutes , and sugar substitutes . for further information on these areas as the us population ages , and more teeth are retained , there will be a higher prevalence of root caries and untreated dental decay . therefore , the demand for dental services in the population of the oldest elderly people is likely to increase . the evaluation of a cohort of elderly aged 79 years or older ( mean age 85.1 years ) with a mean of 19.4 remaining teeth showed that nearly all subjects ( 96 percent ) had coronal decay experience and nearly two - thirds ( 64 percent ) of the individuals had root caries experience , with 23 percent having untreated root caries . utilization of dental services was high among the dentate elderly , with nearly three - quarters reporting having visited a dentist within the past year . those with active coronal or root decay are more likely to be male and to have a history of tobacco use ; they are less likely to have visited a dentist within the past year or report regular use of dental services . the most recent look at caries frequency clearly indicates a marked increase in the prevalence of caries . there are a wide variety of risk factors associated with the development of caries , and although there are differences of opinion regarding the cause of the increase in caries it should be agreed upon that public health strategies are needed to renew the fight against caries and promote prevention of future oral disease . awareness and promotion of water fluoridation , fluoride applications , emphasis on proper tooth brushing with a fluoride dentifrice , flossing , a proper diet , and regular dental office visits can hinder the progression of future caries and can result in an increase in the oral health of all individuals . more programs such as school oral health educational programs are needed to benefit and enhance the oral health ( and systemic health ) of individuals worldwide .

aims and objectives : the purpose of this study was to estimate and assess any correlation between random capillary blood glucose ( rcbg ) and unstimulated whole salivary glucose ( uwsg ) , as well as to estimate various salivary parameters , such as flow rate , ph , buffering capacity , and the influence of these factors on the oral health status in type 2 diabetes mellitus ( dm).materials and methods : sixty individuals suffering from type 2 dm and 40 healthy individuals in the age group of 3060 years were included in the study . rcbg was estimated using glucometer and uwsg was estimated using photocolorimeter . salivary parameters such as flow rate , ph , and buffering capacity were assessed using gc saliva kit . oral health status was recorded using the russell 's periodontal index ( rpi ) and the decayed missing filled teeth ( dmft ) index . the statistical package for the social sciences version 16 was used for statistical analysis.results:type 2 diabetics had higher mean values for rcbg levels and uwsg . type 2 diabetics had low mean salivary flow rate , ph , and buffering capacity . type 2 diabetics had higher mean values for rpi.conclusion:among the salivary factors studied , salivary glucose significantly influenced the periodontal status in type 2 diabetics . root caries is one of the most significant dental problems among older adults today . many studies have demonstrated that older adults are at greater risk for developing root caries . here we examine what risk factors older adults are prone to and explain how they contribute to higher rates of oral disease , in particular root caries . the elderly are at risk for root caries due to dentures , lack of dexterity , a shift from complex to simple sugars , and poor oral hygiene . decreased salivary flow and its manifestations with other social / behavioral and medical factors may provide a more comprehensive explanation to a higher frequency of root caries in older adults .

overweight and obesity is an increasing problem globally . the prevalence is often estimated by body mass index ( bmi ; weight in kg divided by squared height in meters ) . in epidemiological surveys , height and weight are often gathered using self - report questionnaires . this type of data collection is advantageous because it is cost - effective , rapid , and easy to administer when sampling large numbers of individuals , particularly when spread over large areas.1 however , the validity of self - reported data for body measurements has been questioned because some participants overestimate height and underestimate weight , resulting in a lower estimated bmi.15 a review including 64 studies published between 1979 and 2005 examined the validity of self - reported bmi and found that the mean errors varied , and the discrepancies were greatest in obese populations.1 weight status has been shown to predict misreporting of bmi in several large studies , with overweight and obese persons being more likely to under - report bmi.2,610 women have a greater tendency to underestimate bmi than men in many studies,9,11,12 but not all.2 age has been found to predict misreporting of weight and height , with younger women under - reporting weight more than older women , while the chance of over - reporting height increases with age.6,7,11,12 however , these observed associations of age with misreporting are not universal.5 the amount of discrepancy between self - reported bmi and measured bmi varies between studies , and average discrepancies of 0.670.80 kg / m among women have been reported.2,59 even though most studies find relatively small differences between self - reported and measured bmi,8,9 there is a risk that even small deviations can lead to misclassification of bmi and incorrectly estimate associations between bmi categories and incidence of disease.10 the world health organization classifies bmi into four categories : underweight , bmi < 18.5 kg / m ; normal weight , bmi 18.524.9 kg / m ; overweight , bmi 2529.9 kg / m ; and obese , bmi 30 kg / m.13 inaccurate reporting of weight and height may also result in incorrect estimates of the prevalence of overweight . for large studies covering geographically scattered participants , self - reported data are paramount , and examinations of the validity of these data are central to interpretation of the results . validity is a feature of the source population , and can not be inferred from studies from other countries or groups.14 it might change over time and as social and cultural norms about the phenomenon changes.1 to the best of our knowledge , the validity of self - reported height and weight has not been described in a norwegian population . the purpose of this study was to examine whether self - reported weight and height in a sample of women from a large norwegian cohort are valid indicators of bmi for use in epidemiological studies . the norwegian women and cancer ( nowac ) study is a large nationally representative prospective cohort study of women aged 3070 years at recruitment . started in 1991 , the original purpose of the nowac study was to explore the relationship between oral contraceptive use and breast cancer , and the cohort currently includes over 172,000 participants ( figure 1 ) . the study is described in detail by lund et al.15 during 20032006 , the nowac study collected blood samples and a new questionnaire ( hereafter referred to as the secondary questionnaire ) from recent participants born between 1943 and 1957 . the blood collection took place via mail , and the women were asked to contact their general practitioners for taking the blood sample . we focused on a random subsample of 4,498 of the women who were contacted to donate a sample of blood , and therefore were subject to a second data collection within a relatively short time span . of the women contacted , 71% complied ( n=3,194 , figure 1 ) . to limit the time between repeated questionnaires , this study includes the 1,837 women from the random subsample who were asked for and provided a blood sample within 1 year of their primary questionnaire . the primary questionnaire included questions on hormonal and reproductive factors , smoking , alcohol , diet , height , weight , physical activity , self - reported health and some diseases and medications , socioeconomic factors , and sun tanning habits . women were also asked to complete an additional two - page questionnaire , which accompanied the blood sample . this secondary questionnaire collected information that could influence biomarker measurements and updated some of the information from the primary questionnaire , eg , fasting status , menstruation / menopausal status , smoking , height , weight , use of dietary supplements , and medications taken in the previous 7 days . height and weight were self - reported in the primary questionnaire , but could be either self - reported or measured at the secondary questionnaire , and the participants were asked to indicate whether they had been measured by medical staff on the day of the blood sample . no instructions were given with regard to clothing or weight calibration to the participants or medical staff . values producing differences between the two measurements of more than 10 cm for height and 5 kg for weight were checked for data entry errors , but values entered on the original paper questionnaires were assumed to be accurate . missing values for the anthropometric measurements were rare in the primary and secondary questionnaires , with 3% missing for weight and 2% missing for height observed for each questionnaire . there appeared to be a slight tendency to round to the nearest 5 kg for self - reported weight measurements when comparing the frequency of weights ending in 0 or 5 in the primary questionnaire ( 35% ) and secondary questionnaire ( 31% ) , compared with weights measured by medical staff ( 25% ) . bmi was grouped into four categories according to the world health organization classification.13 information about age was based on birth year and provided by the national population register at statistics norway . education , marital status , and perceived health were reported as part of the nowac study . total years of education were categorized into basic or primary education ( 9 years ) , secondary school ( 1012 years ) , and college educated ( 13 years ) . marital status was categorized into married / living together , widowed or divorced , and unmarried . perceived health ( do you think of your own health as : ) was reported as very good , good , poor , or very poor . because few women in the blood sampling group reported very poor health ( n=3 ) equality of group means was tested with two - sample t - tests with pooled variance , while categorical variables were tested using a chi - square test of independence . statistical analyses were performed in sas version 9.4 ( sas institute , cary , nc , usa ) , and statistical significance was defined as a test resulting in a p - value less than 0.05 . repeated measurements of height , weight , and bmi were tested for equality using paired t - tests with pooled variance . differences between self - reported values on the primary questionnaire and values measured by medical staff when blood was sampled ( secondary questionnaire ) represent both changes over time and error . differences between repeated self - reported values are an estimate of changes in weight over time , assuming reporting bias is constant within 1 year . two - sample t - tests with pooled variance were used to test if the differences between types of measurements ( self - reported followed by medical staff - reported ) were larger than repeated self - reported measurements over time . the difference in differences since bmi is often categorized when used as a risk factor , categories were created for both measurements and cross - classified . the percentage correctly classified was calculated , and the agreement between repeated bmi classifications was assessed using a weighted cohen s kappa coefficient.16 this coefficient measures the agreement beyond what is expected by chance . the weighting penalizes errors based upon the level of disagreement ; for example , a misclassification of two categories has a greater penalty than a misclassification of only one category . altman analysis was used to compare the agreement between bmi based on self - reporting and values measured by medical staff , as well as between the two self - reported measurements.17 the average difference between the bmi values indicates the overall bias present in the data , while the limits of agreement ( mean 1.96 standard deviation ) indicate the precision of the measurements . for the comparison with values measured by medical staff , negative differences indicate that the bmi using medical staff values was higher than the self - reported bmi values , ie , negative differences indicate under - reporting . in order to examine representativeness , a comparison was made of basic demographic variables between the study sample group and the independent set of women remaining in the eligible cohort . specifically , the group of women who donated blood within a year of their questionnaire and supplied information on their anthropometric measurements ( n=1,723 ) were compared with those in the eligible cohort , who were willing to donate a blood sample , minus the random subsample ( n=91,828 ) using chi - square tests of independence and two sample t - tests . participants received mailed information about the study together with the questionnaires , and indicated their consent to participate . the study received approval from the regional committee for medical research ethics for the basic collection and storing of questionnaire information and blood sampling . equality of group means was tested with two - sample t - tests with pooled variance , while categorical variables were tested using a chi - square test of independence . statistical analyses were performed in sas version 9.4 ( sas institute , cary , nc , usa ) , and statistical significance was defined as a test resulting in a p - value less than 0.05 . repeated measurements of height , weight , and bmi were tested for equality using paired t - tests with pooled variance . differences between self - reported values on the primary questionnaire and values measured by medical staff when blood was sampled ( secondary questionnaire ) represent both changes over time and error . differences between repeated self - reported values are an estimate of changes in weight over time , assuming reporting bias is constant within 1 year . two - sample t - tests with pooled variance were used to test if the differences between types of measurements ( self - reported followed by medical staff - reported ) were larger than repeated self - reported measurements over time . the difference in differences provides an estimate of any self - report bias . since bmi is often categorized when used as a risk factor , categories were created for both measurements and cross - classified . the percentage correctly classified was calculated , and the agreement between repeated bmi classifications was assessed using a weighted cohen s kappa coefficient.16 this coefficient measures the agreement beyond what is expected by chance . the weighting penalizes errors based upon the level of disagreement ; for example , a misclassification of two categories has a greater penalty than a misclassification of only one category . altman analysis was used to compare the agreement between bmi based on self - reporting and values measured by medical staff , as well as between the two self - reported measurements.17 the average difference between the bmi values indicates the overall bias present in the data , while the limits of agreement ( mean 1.96 standard deviation ) indicate the precision of the measurements . for the comparison with values measured by medical staff , negative differences indicate that the bmi using medical staff values was higher than the self - reported bmi values , ie , negative differences indicate under - reporting . in order to examine representativeness , a comparison was made of basic demographic variables between the study sample group and the independent set of women remaining in the eligible cohort . specifically , the group of women who donated blood within a year of their questionnaire and supplied information on their anthropometric measurements ( n=1,723 ) were compared with those in the eligible cohort , who were willing to donate a blood sample , minus the random subsample ( n=91,828 ) using chi - square tests of independence and two sample t - tests . participants received mailed information about the study together with the questionnaires , and indicated their consent to participate . the study received approval from the regional committee for medical research ethics for the basic collection and storing of questionnaire information and blood sampling . the study sample consisted of 1,837 women who donated blood within a year of their primary questionnaire . of those , 280 women had their height and weight measured by medical staff at the time of their blood sample ( ie , secondary questionnaire ) . the rest either self - reported their height and weight ( n=1,443 ) or failed to indicate how the measurement was taken ( n=114 ) . women who self - reported their weight and height in the secondary questionnaire were similar to those who had their measurements taken by medical staff in terms of demographic characteristics ( table 1 ) . the women had an average age of 53.7 years at the time of their primary questionnaire , with a range of 4664 years . on average , they donated the blood sample 8 months after their primary questionnaire , with a range of 50365 days . comparisons of mean age and days between measurements failed to find any differences between those who self - reported anthropometric measurements at the secondary questionnaire and those who had their measurements taken by medical staff . there were no statistically significant differences between the two groups in terms of education level , marital status , or bmi category from the primary questionnaire . women who self - reported their weight and height at both questionnaires were more likely to have reported very good health status on the primary questionnaire than those who had their values measured at the secondary questionnaire ( p=0.05 ) , but both groups had few women reporting poor health . at the secondary measurement , the prevalence of the different bmi categories were : underweight 1% ( both groups ) , normal weight 51% in the self - reported group versus 49% in the measured group , overweight 34% ( self - reported ) versus 35% ( measured ) , and obesity 14% ( self - reported ) versus 15% ( measured ; see data in tables 3 and 5 ) . the majority of the study sample ( 79% ) self - reported their height and weight at the secondary questionnaire . the repeated self - measurements provide an estimate of changes over time , assuming any bias in reporting remains equal over the time period . on average , women reported little variation in their weight and height , with average differences of 0.6 kg and 0.1 cm ( table 2 ) . only 10% of women reported a weight difference of more than 5 kg ( gain or loss ) and only 10% reported a height difference of more than 1 cm . the mean differences in weight and bmi , although small in value , were statistically significant . for the women who self - reported values on both occasions ( n=1,380 ) , 11% ( n=152 ) were assigned a different bmi category at the secondary questionnaire when compared with the primary questionnaire ( table 3 ) , with 99% ( n=150 ) changing only one category . of those who changed category , the majority ( n=99 , 65% ) increased in bmi . when examining the women who changed bmi category either upward or downward , there were no statistically significant differences . women who increased a bmi category did not differ from those who were classified in the same category on both questionnaires in terms of education level , marital status , perceived health status , mean age , or mean days between measurements . however , there was a tendency for women who moved to a lower bmi category to report poorer health status ( 13% poor , 23% very good ) when compared with those who did not change in bmi category ( 7% and 34% respectively , p=0.07 ) . the normal weight bmi group at the secondary questionnaire had the highest percent agreement with the previously calculated bmi ( 95% ) , followed by the overweight ( 83% ) and obese ( 80% ) groups . few women had bmi values that corresponded to the underweight category . when examining the baseline classification ( row percentages ) in table 3 , there was not much variation with regard to how many women stayed in the same bmi category at the secondary questionnaire among those who self - reported their weight on both occasions , ie , 91% among the normal weight , 86% among the overweight , and 88% among the obese . when donating blood , 15% of the participating women ( n=280 ) had one of the medical staff measure their height and weight for the secondary questionnaire . mean weight as reported by medical staff was on average 1.5 kg higher than that self - reported by women in the primary questionnaire ( table 4 ) . height , on average , was 0.2 cm less than the self - reported values , and bmi calculated from the medical staff values was 0.6 kg / m higher than bmi calculated from self - reported values . while the differences are all small in value , they are statistically significant in paired t - tests . repeated measurements over time can be expected to change , and we estimated these changes for the study sample in the previous section ( table 2 ) . comparisons of the differences in reporting method ( self - reported , measured by medical staff ) and changes over time give an indication of self - report bias . weight measured by medical staff showed a larger increase on average than expected by changes over time ( 0.9 kg ) and the mean difference between reporting methods was significantly larger than that observed between repeated self - reports ( p<0.001 ) . height did not differ significantly more between reporting methods than expected through repeated self - reports . in line with the mean increase in weight , bmi measured by medical staff increased on average by 0.4 kg / m more than observed through repeated self - reports , which is a statistically significant increase ( p=0.002 ) . calculated bmi categories from self - reporting and values measured by medical staff differed for 20% of the women ( n=54 ) by one category ( table 5 ) . of those who changed category , the majority ( n=43 , 80% ) increased in bmi . women who increased one bmi category did not differ from those who were classified in the same category on both questionnaires in terms of education level , marital status , perceived health status , mean age , or mean days between measurements . while the majority of women in the sample were married ( table 1 ) , the 11 women who decreased in bmi category between self - reporting and values measured by medical staff were almost evenly split between married / cohabiting and unmarried ( fisher s exact test , p=0.04 ) . women with normal weight as measured by the medical staff had the highest agreement with the self - reported measure ( 94% ) , followed by obese women ( 80% ) . the highest level of misclassification was among overweight women , where 36% had self - reported values that corresponded to normal weight and 3% had self - reported bmi in the obese range ( table 5 ) . the weighted kappa assessing the agreement between values measured by medical staff and self - reported values was 0.73 ( 95% confidence interval 0.670.80 ) which corresponds to substantial agreement.18 in comparison , the expected agreement was 0.39 ( data not shown ) . when examining the baseline classification ( row percentages ) in table 5 , a slightly different picture emerges . among the self - reported obese , 91% were obese when objectively measured , while among the normal weight ( 78% ) and overweight ( 79% ) , this percentage was lower . in the self - reported normal weight group , most of the misreporters belonged to the overweight category when measured by medical staff ( 21% ) , while the misreporters among the overweight were fairly evenly spread among normal weight and obese when measured by medical staff ( 10% and 11% , respectively ) . the overall mean difference between the average of self - reported bmi and that measured by medical staff was 0.29 kg / m , indicating a small bias toward under - reporting of bmi in self - reported values . the 95% limits of agreement ( figure 2 ) for the differences between the two measurements demonstrate both high overall precision and higher variation between measurements for women in the obese bmi category compared with those in the normal range . women with an average bmi in the obese range were more likely to have under - reported their bmi beyond the 95% limits of agreement ( 18% , 6/33 ) than those with an average bmi in the overweight range ( 5% , 4/82 ) . over the entire range of bmi values , the level of agreement between the two measurements was substantial , as demonstrated by very similar cumulative distribution curves ( figure s1 ) . the under - reporting was slightly greater than that observed for repeated self - reports , which also showed greater variation in the obese bmi range , with 12% of women outside the 95% limits of agreement compared with only 6% outside the limits for the remaining women ( figure 2 ) . there were no statistically significant differences between the eligible cohort and those in the study sample in terms of self - reported weight or perceived health status . there were small but statistically significant differences in terms of mean age at the time of the primary questionnaire ( 53.9 years in the eligible cohort compared with 53.7 years in the study sample p=0.04 ) , marital status ( p=0.04 ) , and height ( 166.8 cm compared with 166.4 cm , p=0.008 ) . women in the study sample were more likely to be married ( 83% ) than those in the eligible cohort ( 81% ) . there was a significant difference between the two groups of women in level of education , with a higher proportion of women in the study sample having a college degree ( 52% ) than those in the comparison group ( 46% , p<0.001 ) . the majority of the study sample ( 79% ) self - reported their height and weight at the secondary questionnaire . the repeated self - measurements provide an estimate of changes over time , assuming any bias in reporting remains equal over the time period . on average , women reported little variation in their weight and height , with average differences of 0.6 kg and 0.1 cm ( table 2 ) . only 10% of women reported a weight difference of more than 5 kg ( gain or loss ) and only 10% reported a height difference of more than 1 cm . the mean differences in weight and bmi , although small in value , were statistically significant . for the women who self - reported values on both occasions ( n=1,380 ) , 11% ( n=152 ) were assigned a different bmi category at the secondary questionnaire when compared with the primary questionnaire ( table 3 ) , with 99% ( n=150 ) changing only one category . of those who changed category , the majority ( n=99 , 65% ) increased in bmi . when examining the women who changed bmi category either upward or downward , there were no statistically significant differences . women who increased a bmi category did not differ from those who were classified in the same category on both questionnaires in terms of education level , marital status , perceived health status , mean age , or mean days between measurements . however , there was a tendency for women who moved to a lower bmi category to report poorer health status ( 13% poor , 23% very good ) when compared with those who did not change in bmi category ( 7% and 34% respectively , p=0.07 ) . the normal weight bmi group at the secondary questionnaire had the highest percent agreement with the previously calculated bmi ( 95% ) , followed by the overweight ( 83% ) and obese ( 80% ) groups . few women had bmi values that corresponded to the underweight category . when examining the baseline classification ( row percentages ) in table 3 , there was not much variation with regard to how many women stayed in the same bmi category at the secondary questionnaire among those who self - reported their weight on both occasions , ie , 91% among the normal weight , 86% among the overweight , and 88% among the obese . when donating blood , 15% of the participating women ( n=280 ) had one of the medical staff measure their height and weight for the secondary questionnaire . mean weight as reported by medical staff was on average 1.5 kg higher than that self - reported by women in the primary questionnaire ( table 4 ) . height , on average , was 0.2 cm less than the self - reported values , and bmi calculated from the medical staff values was 0.6 kg / m higher than bmi calculated from self - reported values . while the differences are all small in value , they are statistically significant in paired t - tests . repeated measurements over time can be expected to change , and we estimated these changes for the study sample in the previous section ( table 2 ) . comparisons of the differences in reporting method ( self - reported , measured by medical staff ) and changes over time give an indication of self - report bias . weight measured by medical staff showed a larger increase on average than expected by changes over time ( 0.9 kg ) and the mean difference between reporting methods was significantly larger than that observed between repeated self - reports ( p<0.001 ) . height did not differ significantly more between reporting methods than expected through repeated self - reports . in line with the mean increase in weight , bmi measured by medical staff increased on average by 0.4 kg / m more than observed through repeated self - reports , which is a statistically significant increase ( p=0.002 ) . calculated bmi categories from self - reporting and values measured by medical staff differed for 20% of the women ( n=54 ) by one category ( table 5 ) . of those who changed category , the majority ( n=43 , 80% ) increased in bmi . women who increased one bmi category did not differ from those who were classified in the same category on both questionnaires in terms of education level , marital status , perceived health status , mean age , or mean days between measurements . while the majority of women in the sample were married ( table 1 ) , the 11 women who decreased in bmi category between self - reporting and values measured by medical staff were almost evenly split between married / cohabiting and unmarried ( fisher s exact test , p=0.04 ) . women with normal weight as measured by the medical staff had the highest agreement with the self - reported measure ( 94% ) , followed by obese women ( 80% ) . the highest level of misclassification was among overweight women , where 36% had self - reported values that corresponded to normal weight and 3% had self - reported bmi in the obese range ( table 5 ) . the weighted kappa assessing the agreement between values measured by medical staff and self - reported values was 0.73 ( 95% confidence interval 0.670.80 ) which corresponds to substantial agreement.18 in comparison , the expected agreement was 0.39 ( data not shown ) . when examining the baseline classification ( row percentages ) in table 5 , a slightly different picture emerges . among the self - reported obese , 91% were obese when objectively measured , while among the normal weight ( 78% ) and overweight ( 79% ) , this percentage was lower . in the self - reported normal weight group , most of the misreporters belonged to the overweight category when measured by medical staff ( 21% ) , while the misreporters among the overweight were fairly evenly spread among normal weight and obese when measured by medical staff ( 10% and 11% , respectively ) . the overall mean difference between the average of self - reported bmi and that measured by medical staff was 0.29 kg / m , indicating a small bias toward under - reporting of bmi in self - reported values . the 95% limits of agreement ( figure 2 ) for the differences between the two measurements demonstrate both high overall precision and higher variation between measurements for women in the obese bmi category compared with those in the normal range . women with an average bmi in the obese range were more likely to have under - reported their bmi beyond the 95% limits of agreement ( 18% , 6/33 ) than those with an average bmi in the overweight range ( 5% , 4/82 ) . over the entire range of bmi values , the level of agreement between the two measurements was substantial , as demonstrated by very similar cumulative distribution curves ( figure s1 ) . the under - reporting was slightly greater than that observed for repeated self - reports , which also showed greater variation in the obese bmi range , with 12% of women outside the 95% limits of agreement compared with only 6% outside the limits for the remaining women ( figure 2 ) . there were no statistically significant differences between the eligible cohort and those in the study sample in terms of self - reported weight or perceived health status . there were small but statistically significant differences in terms of mean age at the time of the primary questionnaire ( 53.9 years in the eligible cohort compared with 53.7 years in the study sample p=0.04 ) , marital status ( p=0.04 ) , and height ( 166.8 cm compared with 166.4 cm , p=0.008 ) . women in the study sample were more likely to be married ( 83% ) than those in the eligible cohort ( 81% ) . there was a significant difference between the two groups of women in level of education , with a higher proportion of women in the study sample having a college degree ( 52% ) than those in the comparison group ( 46% , p<0.001 ) . in this study , we estimated the misreporting of self - reported weight and height by comparing repeated self - reports with self - reported values followed by values measured by medical staff . the two groups did not differ except for better perceived health among those who gave repeated self - reports . the distribution of bmi categories did not differ between self - reported and measured values . weight and bmi were under - reported more by the repeated self - reporting group , but there was substantial agreement between self - reported values and those measured by medical staff ( weighted kappa for bmi 0.73 ) . under - reporting leading to misclassification of bmi category was most common among overweight women ( 36% ) , but the highest proportion of extreme under - reporters was found in the obese women ( 18% outside the 95% limits of agreement ) . the highest proportion of correctly classified women based on the primary self - reported measurement was found among the obese women . although the design of this validation study differs from that of most other studies , our findings are in accordance with studies comparing self - reported weight and height with weight and height measured by medical staff within a short time period . the difference in self - reported bmi and bmi measured by medical staff in our study was small but statistically significant , ie , 0.4 kg / m , and lower than what was found in women in the adventist health study ( 0.7 kg / m),2 epic - norfolk ( 0.92 kg / m),19 multiethnic cohort ( 0.67 kg / m),8 the sister study ( 0.7 kg / m),6 and in the skaraborg project ( 0.8 kg / m),7 women attending a us family medicine clinic ( 0.8 kg / m),5 and female participants in the national health and nutrition education survey iii ( 0.67 kg / m).9 the results were similar to those found for women in an austrian study ( 0.43 kg / m),20 but the difference was larger than what was found in australian women ( 0.12 kg / m).21 an earlier review found mean differences between self - reported and measured bmi of 0.9 to 1.2 kg / m in women from the general population.1 as in most other studies , the errors in bmi were due to under - reporting of weight , and there was no significant misreporting of height . in the national health and nutrition education survey iii , high correlations was found between self - reported and measured bmi , and also between self - reported and measured bmi and disease biomarkers.9 the correlations did not differ much by age , sex , or obesity status . further , the results were the same when the analyses were done with percent body fat rather than bmi as the measure of adiposity . stommel et al found that women aged 4255 years reported bmi more in accordance with direct measures than either their younger or older counterparts,22 and most of our participants were in that age range . a more recent paper found that there have been temporal changes in the precision of self - reported height and weight , leading to more accurate bmi estimations.23 as the opposite has also been found,24 there could be cultural differences in accuracy of self - reported anthropometric values , but we have not found other norwegian publications for comparison . correlations between self - reported and measured bmi are generally high ( > 0.90 in all ethnic groups9 ) , but they are not adequate for measuring reliability , because they are testing associations rather than agreement , and are not able to identify systematic errors.25 the percentage of agreement includes both the precision of the measurement and the frequency of errors , and has clinical meaning , while graphical presentations are useful for displaying distributions and the magnitude of error.25 in general , substantial agreement between bmi categorizations based on self - reporting and measured values has been found , but self - reported values tend to give a lower bmi category,1,2,7,11,20 especially in overweight and obese subjects.3,4,12,26,27 kappa values between 0.66 and 0.81 have been reported,2,12,19,21 and our value of 0.73 fits well with this . in our study , 80% of the women were correctly classified , similar to what others have found.2,5,21 studies vary as to whether the percentage of correctly classified women is lowest among the overweight5,21 or obese.68,12,19,20 in our study , the percentage was lowest among the overweight when the underweight group ( n=2 measured by medical staff ) was disregarded . when examining those who were measured by medical staff based on their self - reported bmi category at baseline , the highest percentage of correctly classified participants was found in the obese group ( 91% ) . hence , the lower percentage of correctly classified obese women based on the measured values was due to misclassification ( under - reporting ) among those who were self - reported overweight rather than misclassification ( over - reporting ) among the obese . the repeated self - reported values indicated a greater range of values for those with an average bmi in the obese range compared with other women . this may indicate errors in measurement or changes in reporting bias , but could also indicate greater variation in weight over time for women in the obese range . in a large public health study from spain , after adjusting for predictors of under - reporting of bmi , especially dissatisfaction with body size , the estimated overweight prevalence increased from 15.0% to 18.5%.3 a large validity study of self - reported bmi in the national health and nutrition education survey concluded that self - reports are sufficient for most epidemiological studies , but not for prevalence studies.9 in our study , the prevalence of overweight and obesity did not differ when using self - reported and measured values . since the validation subsample was representative , and there was no difference between the self - reported and measured group , except in perceived health status , it seems that the self - reported values may be utilized for estimating prevalence of overweight and , in particular , obesity . comparisons failed to find any differences between those whose anthropometric measurements were self - reported in the secondary questionnaire and those who were measured by medical staff , except that women who gave repeated self - reports were more likely to report very good health status on the primary questionnaire than those who had their values measured for the secondary questionnaire . it is likely that perceived health influences what kind of questions a woman asks the medical staff ( blood sample only , or also anthropometric measurements ) . also , women who perceived their health as good might not be interested in receiving feedback from medical staff or in spending any additional time in the office . there was no difference in the prevalence of self - perceived poor health between the two groups . we have previously shown that participants in the nowac study are representative of the female norwegian population as a whole,28 except for higher education than non - responders , and that cancer rates are the same in our cohort as in the general female population of the same age.15 in the current study , education was the only factor where significant differences were found between women who gave blood and the remaining cohort . there were no significant differences in bmi between the different education groups , so the validity of the present study is not threatened . the strengths of this study include the representativeness of the study sample and the unbiased study design . participants were not aware that their anthropometric measurements might be checked when providing their primary height and weight information . knowledge of future measurement of weight by medical staff may lead to more accurate reporting.27 this study has some limitations . different measurement instruments were used for different participants ; they were not calibrated nor were precise instructions provided . participants with values measured by medical staff would have used a different scale and measuring tape at home when providing the primary self - reported values . this variation in instruments undoubtedly increased the variability in the measurements and the measurement error . failure to calibrate instruments has been shown to increase the prevalence of overweight and obesity in population - based samples.29 however , digital home bathroom scales have been shown to provide sufficiently accurate and consistent weights for public health research purposes.30 further , the mean time lapse between measurements was quite long ( 8 months ) , but as the difference was equal in both groups , this complicates only the study design and not the final results . body weight may naturally shift up and down over time , so we studied differences in excess of what was found with repeated self - reports , assuming constant misreporting over the year . this could be questioned , but since the data collection took place over years , the results should be robust to seasonal variations . the results of this study show that self - reported data successfully distinguish between the obese and nonobese , and although there were some more misreporters among the overweight , the results are comparable with those of other studies . being able to correctly classify the obese is important , given that the association between bmi and mortality or morbidity is strongest for this group.31,32 women who had their weight measured after having self - reported had a significantly higher weight than those who self - reported twice . the tendency of under - reporting was largest among overweight women , while the most extreme under - reporters were found in the obese group . despite the under - reporting , the discrepancies between self - reported and directly measured bmi in women were small , and the agreement between self - reported and measured values was substantial , as demonstrated by the cumulative distribution of the bmi curves . our self - reported weight and height data provide a valid ranking of bmi for middle - aged norwegian women . cumulative distribution plot of self - reported and measured bmi . abbreviation : bmi , body mass index . the functions of the salivary glands are controlled by the autonomic nervous system and influenced by the sensory nervous system . when parasympathetic impulses dominate , salivary flow is greatly enhanced and the saliva has a low protein content . studies of animal and human innervation have revealed that parasympathetic nerve fibers are present around acinar cells , ducts , and blood vessels in the major salivary glands . a research has also shown that beside the classic transmitters noradrenaline and acetylcholine , neuropeptides such as substance p ( sp ) , calcitonin gene - related peptide ( cgrp ) , and vasoactive intestinal polypeptide ( vip ) ( figure 1 ) are present in the nerve fibers of the autonomic nervous system as well as in the auriculotemporal nerve , facial nerve , and cervical dorsal root fibers . these neuropeptides are known to cause salivation in rats [ 27 ] . in recent years , the mechanisms of actions of drugs that used to treat xerostomia have been elucidated pharmacologically from the viewpoint of salivary neuropeptide levels . anethole trithione and pilocarpine have been shown to elevate sp and cgrp levels in human saliva [ 811 ] . cevimeline hydrochloride hydrate ( cevimeline ) ( figure 2 ) is a novel muscarinic acetylcholine receptor agonist currently being developed as a therapeutic agent for sjgren 's syndrome . sjgren 's syndrome is a serious and chronic autoimmune disorder characterized by inflammation in the exocrine glands such as the salivary and lacrimal glands , leading to xerostomia ( dry mouth ) and xerophthalmia ( dry eyes ) . cevimeline acts as a stimulator of the m3 acetylcholine receptor expressed on salivary glands and has been shown to increase saliva secretion in patients with sjgren 's syndrome . although cevimeline is useful for the treatment of dry mouth , it only enhances saliva production in 60% of the patients , and the mechanism of the drug response is still unknown . it is possible that individual variability of neuropeptide nerve stimulation in response to cevimeline may be involved in the variable drug response to cevimeline . the objective of the present study is to examine the effects of cevimeline on saliva and plasma levels of sp- , cgrp- , and vip - like immunoreactive substances ( iss ) in humans , as markers of nerve stimulation of these neuropeptides . cevimeline hydrochloride hydrate ( saligren capsule 30 mg ) was purchased from nippon kayaku co. ltd . lactose ( merck hoei co. ltd . , osaka , japan ) was used as placebo . synthetic human sp , cgrp and its fragment ( 837 ) , and vip were purchased from peptide institute , inc . vip fragment ( 1128 ) was supplied by professor yajima ( kyoto university , kyoto , japan ) . substance p antiserum ( y150 ) was purchased from yanaihara institute ( shizuoka , japan ) , cgrp antiserum ( 14160 ) from peptide institute , inc . , and vip antiserum ( t-4116 ) from peninsula laboratories ( california , usa ) . seven healthy nonsmoking male volunteers aged 2431 ( median 27 ) years and weighing 5670 ( median 64 ) kg participated in this study . all subjects had no history of xerostomia , and their baseline fasting salivary and plasma levels of sp- , cgrp- , and vip - is were within the normal ranges for healthy subjects reported previously [ 811 , 15 , 16 ] . each subject gave informed consent after receiving explanation on the scientific purpose of the study . the subjects fasted for at least 2 hours before the study was commenced and during the experiments . we performed an open - labeled , crossover study between may and october 2010 . in each subject , cevimeline and placebo were studied in random order , in a crossover manner with an interval of one month between the two studies . on the day of study , all subjects finished lunch ( standardized lunch of less than 800 kcal ) before 12:00 . each study was conducted from 14:00 to 18:00 in a room with temperature controlled at 25c , during which the subjects maintained a resting and relaxed state . a single dose of cevimeline 30 mg ( cevimeline group ) or placebo ( placebo group ) was administered orally with 100 ml water . at scheduled times after the test drug was administered , saliva production was measured , and saliva samples were collected for assaying salivary neuropeptide levels , while blood samples were collected for measuring plasma neuropeptide levels . the dose of cevimeline in this study was the normal daily dose used in clinical therapy . saliva and venous blood samples were collected before and at 30 , 60 , 90 , 120 , 180 , and 240 min after administration of cevimeline or placebo . the volume of saliva produced in 5 min was measured by the saxon test , an oral equivalent of the schirmer test . 14 , kawamoto houtai zairyou , osaka , japan ) and a polyethylene pouch were weighed . after swallowing to remove any existing oral fluid , saliva was collected by placing the two cotton balls onto the vestibule of the mouth for exactly 5 min . the weight of saliva was determined by subtracting the original weight of the pouch and cotton balls from the weight obtained after the cotton balls were placed in the mouth . the weight of the liquid was taken to be the salivary volume ( ml ) produced in 5 minutes . unstimulated whole saliva specimens were collected by the spitting method according to navazesh and christensen . the subjects rinsed their mouth thoroughly with deionized water and rested for a few minutes before saliva collection began . after one minute practice collection , which was discarded , subsequently 3 ml of saliva was collected into a test tube containing 500 kallikrein inhibitor units / ml of aprotinin and 1.2 mg / ml of edta . blood samples were collected into chilled tubes containing 500 kallikrein inhibitor units / ml of aprotinin and 1.2 mg / ml of edta . the saliva samples were diluted 1 : 1 with 4% acetate buffer ( ph 4.0 ) , centrifuged at 3500 rpm for 5 min at 4c , and then the supernatant was diluted 2 : 3 with 4% acetate buffer ( ph 4.0 ) and loaded onto c18 reverse - phase cartridges ( sep - pak c18 ; millipore corp . , milford , ma , usa ) . blood samples were centrifuged , and the plasma samples were diluted 1 : 4 with 4% acetate buffer ( ph 4.0 ) and loaded onto c18 reverse - phase cartridges . after washing with 4% acetate buffer , neuropeptides in the columns were eluted with 70% acetonitrile in 0.5% acetate buffer ( ph 4.0 ) . eluates were concentrated by spin - vacuum evaporation , lyophilized , and stored at 40c until use . the recovery of sp- , cgrp- , and vip - is in saliva and plasma was greater than 90% using this extraction procedure [ 1921 ] . neuropeptide levels in saliva and plasma were measured using highly sensitive enzyme immunoassays for sp , cgrp , and vip as described previously . an immunoplate ( nunc - immuno module maxisorp f8 , intermed , denmark ) coated with anti - rabbit igg ( 55641 , icn pharmaceuticals , inc . , human sp , cgrp fragment ( 837 ) , or vip fragment ( 1128 ) was conjugated with -d - galactosidase by n-(-maleimido - caproyloxy)-succinimide according to the methods of kitagawa et al . . the enzyme immunoassays were specific and highly sensitive , with detection limits of 0.08 , 0.40 , and 1.00 fmol / well for sp- , cgrp- , and vip - is , respectively . total release of each neuropeptide or saliva was calculated as the area under the level or volume time curve ( auc0240 ) using the trapezoidal method . differences in neuropeptide - is level , salivary volume , and their auc0240 between the cevimeline and placebo groups were analyzed by paired t - test or mann - whitney u test . the relationship between auc0240 of neuropeptide - is level and auc0240 of salivary volume was analyzed by pearson 's product - moment correlation coefficient . statistical analyses were performed using the spss software package ( version 17.0 ; spss inc . , il , usa ) . the salivary sp - is level - time profile and total release of sp - is ( auc0240 ) after a single oral dose of cevimeline or placebo are shown in figure 3(a ) and table 1 . oral administration of cevimeline resulted in significant increases in salivary sp - is level at 30 , 60 , 90 , and 120 min ( 7.5 3.4 , 19.1 15.1 , 12.5 5.1 , and 9.9 4.1 pg / ml , resp . ) compared with the corresponding levels after placebo administration ( 4.0 1.5 , 5.2 1.8 , 5.6 2.4 , and 5.3 2.7 pg / ml ) . furthermore , auc0240 was significantly higher after cevimeline administration ( 2420.9 744.6 pgmin / ml ) compared with placebo ( 1185.8 398.6 pgmin / ml ) . on the other hand , no significant changes in salivary cgrp- and vip - is levels and auc0240 were observed after the administration of cevimeline ( figures 3(b ) and 3(c ) and table 1 ) compared with placebo . the plasma sp- , cgrp- , and vip - is level - time profiles and total releases of sp- , cgrp- , and vip - is ( auc0240 ) after a single oral dose of cevimeline or placebo are shown in figure 4 and table 2 . cevimeline administration did not alter the plasma levels or auc0240 of sp- , cgrp- , or vip - is compared with placebo . the changes in salivary volume and total release of saliva ( auc0240 ) after cevimeline or placebo administration are shown in figure 5 and table 3 . cevimeline administration resulted in significant increases in salivary volume at 90 , 180 , and 240 min ( 5.6 2.8 , 5.7 1.8 , and 5.1 1.2 ml , resp . ) compared with the corresponding levels after placebo administration ( 3.4 1.3 , 3.4 1.5 , and 3.2 1.6 ml ) . the auc0240 was also significantly higher after cevimeline administration ( 1200.8 403.4 mlmin ) compared with placebo ( 804.9 369.8 mlmin ) . the relationship between auc0240 of sp - is level and salivary volume after administration of cevimeline or placebo is shown in figure 6 . a significant correlation was observed between auc0240 of sp - is level and auc0240 salivary volume ( r = 0.55 , p = 0.042 ) . in this study , we investigated the effects of cevimeline on saliva and plasma levels of sp- , cgrp- , and vip - is in healthy subjects . past studies have established that salivary and plasma levels of sp- , cgrp- , and vip - is vary within 30 min after a meal and then maintain constant from 1 hour after a meal [ 8 , 9 , 1921 ] . furthermore , it is known that the absorption of cevimeline is little affected by a meal . these data support our study design , and the present study appropriately evaluates the effects of cevimeline on neuropeptide levels and saliva production without being affected by a meal . sp is mainly localized in submandibular and parotid glands and increases blood flow via its vasodilatory effect in salivary glands , stimulates the production of saliva and amylase , and influences ionic flow in rats [ 23 , 24 ] . previous report indicates that cgrp also enhances the release of saliva and amylase in rats [ 3 , 6 ] , and vip induces alterations in salivary fluid and protein secretion [ 4 , 25 ] . in the present study , a single oral dose of cevimeline resulted in significant increases in salivary sp - is level at 30 , 60 , 90 , and 120 min and in the auc0240 of sp - is compared with placebo administration , whereas cevimeline did not alter the plasma levels or auc0240 of sp - is . anethole trithione and pilocarpine have also been reported to increase sp - is in saliva but not in plasma [ 811 ] . these results indicate a close association of sp with the enhancement of salivary secretion by cevimeline , in the same manner as anethole trithione and pilocarpine . in addition , these findings suggest that cevimeline may mainly promote salivary secretion from submandibular and parotid glands by increasing sp . on the other hand , no significant changes in salivary and plasma levels and auc0240 of cgrp- and vip these findings suggest that pathways via cgrp and vip nerves may not be involved in the stimulatory effect on salivation by cevimeline . on the other hand , anethole trithione and pilocarpine increase not only sp but also cgrp levels in human saliva [ 811 ] . cevimeline acts as a selective stimulator of the m3 acetylcholine receptor expressed on salivary glands , and this selectivity may reflect the specificity of the cevimeline action on sp nerves in salivary glands . oral cevimeline administration resulted in significant increases in salivary volumes at 90 , 180 , and 240 min and in the auc0240 compared with placebo administration . furthermore , a significant correlation was observed between auc0240 of sp - is level and auc0240 of salivary volume , suggesting the possible involvement of sp in the cevimeline - enhanced saliva secretory activity . a lag time was observed between elevation of salivary sp level and increase in salivary volume , suggesting that sp secreted from the sp nerves stimulated by cevimeline may initially increase blood flow and cause vasodilatation in salivary glands , followed by a gradual increase in salivary production . however , some reports have suggested that human salivary glands are thought to lack an sp innervation of the acinar cells , and in vitro pieces of human submandibular glands do not respond with fluid secretion to the administration of sp , as judged by the release of potassium [ 26 , 27 ] . furthermore , other neuropeptides not tested in this study may also be involved in the mechanism of enhancement of salivary secretion by cevimeline . therefore , this notion requires verification by further studies . cevimeline is known to enhance saliva production in only 60% of the treated patients , and the mechanism of drug response remains unknown . the present study shows a possibility that individual variability of sp nerve stimulation in response to cevimeline may account for the variable drug response to cevimeline , although it is uncertain whether this trend in healthy volunteers is also observed in patients . therefore , further studies are required to investigate the effects of cevimeline in patients with conditions such as xerostomia . this study demonstrated the effects of cevimeline on salivary and plasma levels of neuropeptides in humans . a single oral dose of cevimeline resulted in a significant increase in salivary but not plasma sp - is level , and a significant correlation was observed between the total release of salivary sp - is and of salivary volume . these findings suggest a close association of sp with the enhancement of salivary secretion by cevimeline . a large - scale controlled study evaluating multiple dosing conditions of cevimeline would help to better understand the effects of cevimeline .

backgroundbody mass index ( bmi ) based on self - reported height and weight has been criticized as being biased because of an observed tendency for overweight and obese people to overestimate height and underestimate weight , resulting in higher misclassification for these groups . we examined the validity of bmi based on self - reported values in a sample of norwegian women aged 4464 years.methodsthe study sample of 1,837 participants in the norwegian women and cancer study self - reported height and weight , and then , within 1 year , either self - reported anthropometric again , or were measured by medical staff . demographic and anthropometric were compared using t - tests and chi - square tests of independence . misclassification of bmi categories was assessed by weighted cohen s kappa and bland altman plot.resultson average , the two measurements were taken 8 months apart , and self - reported weight increased by 0.6 kg ( p<0.05 ) , and bmi by 0.2 kg / m2 ( p<0.05 ) . the distribution of bmi categories did not differ between self - reported and measured values . there was substantial agreement between self - reported values and those measured by medical staff ( weighted kappa 0.73 ) . under - reporting resulting in misclassification of bmi category was most common among overweight women ( 36% ) , but the highest proportion of extreme under - reporting was found in obese women ( 18% outside the 95% limits of agreement ) . the cumulative distribution curves for the measured and self - reported values closely followed each other , but measurements by medical staff were shifted slightly toward higher bmi values.conclusionwhile there was substantial agreement between self - reported and measured bmi values , there was small but statistically significant under - reporting of weight and thus self - reported bmi . the tendency to under - report was largest among overweight women , while the largest degree of under - reporting was found in the obese group . self - reported weight and height provide a valid ranking of bmi for middle - aged norwegian women . cevimeline is a novel muscarinic acetylcholine receptor agonist currently being developed as a therapeutic agent for xerostomia . we examined the effects of cevimeline on salivary and plasma levels of substance - p- ( sp- ) , calcitonin - gene - related - peptide- ( cgrp- ) , and vasoactive - intestinal - polypeptide- ( vip- ) like immunoreactive substances ( iss ) in humans . an open - labeled crossover study was conducted on seven healthy volunteers . saliva volume was measured , and saliva and venous blood samples were collected before and 30240 min after a single oral dose of cevimeline or placebo . salivary and plasma levels of sp- , cgrp- , and vip - is were measured using a highly sensitive enzyme immunoassay . a single oral dose of cevimeline resulted in significant increases in salivary but not plasma sp - is level compared to placebo . cevimeline administration did not alter the salivary or plasma levels of cgrp - is or vip - is compared to placebo . significant increases in salivary volume were observed after cevimeline administration compared to placebo . a significant correlation was observed between the total release of sp - is and that of salivary volume . these findings suggest an association of sp with the enhancement of salivary secretion by cevimeline .

the technique of lymphatic mapping and sentinel lymph node biopsy ( slnb ) has emerged in the last two decades as a minimally invasive approach to evaluate regional lymph node basins in patients with intermediate and high - risk primary cutaneous melanoma . in particular , slnb is now recommended as a staging procedure for patients with t2 , t3 or t4 melanomas and clinical uninvolved regional lymph nodes ( clinical stage ib and ii ) and suggested also for patients with t1 melanomas and pathologic features associated with an increased risk of nodal micrometastases ( ulceration , high mitotic rate , ) . also positron emission tomography ( pet ) with 18f - fluorodeoxyglucose ( 18f - fdg ) has been extensively investigated in patients with melanoma and plenty of studies have shown its effective role in detecting distant metastases , further increased after the introduction of co - registered computed tomography ( ct ) scan ( 18f - fdg pet / ct ) . in this article , we introduce a case of pt4b thigh melanoma , in which both procedures were performed , together with ultrasonography . an 82-year - old white male , with a clinically - confirmed cutaneous melanoma of the right thigh , presented to our unit to undergo lymphoscintigraphy , in order to perform slnb at the same time of tumor excision . lymphoscintigraphy with tc - nanocolloids was performed on a hybrid system philips single - photon emission computed tomography / computed tomography ( spect / ct ) precedence 16 slices ( philips healthcare , eindhoven , the netherlands ) after intradermal injection of the radiopharmaceutical around the primary lesion ( four separate injections , 0.1 ml for each aliquot , total activity 100 mbq ) . low dose helical ct scan was performed : 120 kv , 100 ma , d - dom control dose , 3 mm slice thickness , 1.5 mm detector collimation , pitch 0.8 , rotation time 0.75 s. spect scan was acquired with the following parameters : 128 128 matrix size , 120 view angle , 10 s time / angle , 5 mm pixel size . spect / ct images showed uptake of the radiocolloids in a right inguinal lymph node . on ct co - registered images , anyway , another lymph node with no radiopharmaceutical uptake but with suspicious aspect ( globular morphology , absence of hilum ) was detectable in the crural region , much closer to the primary tumor [ figures 1 and 2 - left panel ] . axial views of single - photon emission computed tomography / computed tomography ( ct ) lymphoscintigraphy with tc - nanocolloids . tracer uptake can be seen in the site of injection / primary tumor ( green arrow ) and in a right inguinal node ( yellow arrow ) , while there is no uptake in a crural node with suspicious aspect on ct images ( red arrow ) volume rendering of technetium - labeled radiocolloids single - photon emission computed tomography / computed tomography ( left panel ) and f - fluorodeoxyglucose positron emission tomography / computed tomography ( right panel ) . the arrows show the sites of primary lesion ( green ) , true sentinel crural node ( red ) , false sentinel inguinal node ( yellow ) for this reason , and due to the adverse pathologic features of the removed lesion ( clark 's level iv , breslow 's depth 4.6 mm , ulceration , 89 mitoses / mm , poor inflammatory infiltrate , pt4b ) , the patient was further staged with a f - fdg pet / ct scan after surgery . pet / ct showed pathologic uptake of the tracer in the suspected right crural lymph node , which was removed : no other nodal or visceral metastases were seen [ figures 2 - right panel and 3 ] . histology demonstrated signs of chronic inflammation and no neoplastic cells in the inguinal lymph node ( analysis of slices from the whole node with hematoxylin and eosin ( h and e ) stain and confirmation with immunohistochemical staining for s-100 protein in each blank slide ) , while a massive metastasis from melanoma was seen in the crural node ( h and e ) . no significant uptake can be seen in the site of the removed primary tumor ( green arrow ) and right inguinal node ( yellow arrow ) , while high metabolic activity is demonstrated in the crural node ( red arrow ) neither inguinal lymphadenectomy nor systemic therapy was proposed , due to age and co - morbidity ( hypertensive cardiomyopathy ) and a follow - up based on abdominal , and inguinal ultrasonography was organized . ten months after surgery the patient developed a metastatic disease , further depicted by a follow - up f - fdg pet / ct scan [ figure 4 ] . follow - up f - fluorodeoxyglucose positron emission tomography / computed tomography scan ( 1 year after tumor excision ) : multiple secondary lesions can be seen in right inguinal nodes , in the liver and in many skeletal segments the prognostic factors for cutaneous melanoma have been recently revised in the 7 edition of the american joint committee on cancer ( ajcc ) ( 2009 ) , based on analysis of data for over 50,000 patients of ajcc database . apart from the features of the primary lesion ( thickness , mitotic rate , ulceration ) , the histologic status of regional nodes has been confirmed as the most powerful independent predictor of survival in clinically node - negative patients . the technique of slnb , first proposed in the 1980s , made inroads once it was clear that the treatment of regional node disease while still microscopic afforded a survival benefit compared to waiting for clinically evident disease . this strategy , minimally invasive , allows the use of more aggressive surgical approaches and systemic therapies only in higher - risk patients , with occult stage iii disease . it has shown high sensitivity ( especially when performed with spect / ct - aided lymphatic mapping and multiple peri - tumour injections ) with very low false - negative rate , mainly related to technical problems associated with identification of the true sentinel node ( sn ) by nuclear medicine physicians and surgeons and errors in tissue sampling and interpretation by pathologists . thus , even if new and more sensitive molecular techniques have already shown promising results , slnb with pathological assessment is now the recommended staging procedure for all stage i and ii patients with primary melanomas > 1.0 mm in thickness . nonetheless , with the introduction and the development of noninvasive metabolic imaging techniques ( such as f - fdg pet / ct ) also in patients affected by melanoma , the hypothesis that even slnb could be avoided and replaced by the analysis of tumor metabolism in the lymphatic basin has emerged . in the last 15 years , almost 20 papers concerning the diagnostic performance of f - fdg pet / ct in comparison to slnb ( and ultrasonography ) can be found in literature , all pointing out , with few exceptions , a very low sensitivity of f - fdg pet / ct in discovering small lymph node metastases if compared with slnb . in this scenario , our report looks somewhat interesting , suggesting the possible utility of pet / ct even in the evaluation of regional disease in selected patients . the key point seems to be the missed identification of sentinel lymph node by lymphoscintigraphy , probably due to the obstruction of lymphatic flow to the true sn and the consequent deviation of the flow to another node . this situation has already been described in some papers about sns in melanoma and also in breast cancer . in 2009 , for example , lam et al . described three cases in which preoperative lymphoscintigraphy failed to show sns containing metastatic melanoma ( all with significant tumor involvement ) , that were discovered by ultrasound and then confirmed by fine - needle aspiration biopsy and histopathology . the same circumstance was previously described by estourgie in 2003 in two breast cancer patients : in that report , both lymphoscintigraphy with tc - nanocolloid and patent blue dye administration failed to identify the true sn , completely invaded by tumor and discovered by intra - operative palpation of the biopsy wound . what seems new here is that not only lymphoscintigraphy , but even preoperative ultrasonography failed to identify the metastatic crural node . in this setting , in our opinion , a double lesson can be learnt . first , this report confirms that a small risk of missing sentinel lymph node by lymphoscintigraphy exists ( especially in thick melanoma ) and highlights the added value of a hybrid tomographic study ( spect / ct ) , that allows a morphologic evaluation of the interested region too . second , it shows that f - fdg pet / ct , usually performed for n staging ( for the well - known lack of sensitivity in the study of the lymphatic basin ) , could give important information also about regional disease in selected patients . the selection of the staging procedures to perform should always be individualized , considering general and local features of the disease , and evaluating together with the patient the risks and benefits of each technique . the chronic diseases like cardiovascular diseases , diabetes and cancer contributes to almost half of the all - cause mortality in india . weighted prevalence of ischemic heart diseases was observed as 25.3/1000 population , and 118.0/1000 population for diabetes mellitus in an urban area of india . it was observed due to common risk factors like physical inactivity , unhealthy diet , tobacco use and stress . along with growth and development , reduction in common risk factors was considered to be one of the essential preventive and control strategies for chronic diseases . like modern medicine , the complementary and alternative medicine ( cam ) - ayurveda ( knowledge or science of life ) - primarily concerned with the body . it had been mentioned in ayurveda , about the three pillars for healthy and long life ; proper diet ( ahar ) , proper activities ( vihar ) , and divine life - style and control of sexuality ( brahmacharya ) . improper and unbalanced diet like undigested , junk , and accumulated food was also considered toxic for the body . ayurveda had been oldest and most organized scientific discipline , which was providing health - care . once upon a time , their classical text like charak samhita ( 100 ad ) is an excellent source of information about the holistic health , herbs , diseases , and surgical treatment for the entire country and the world . like yoga , ayurveda derived its origin to the hoary vedas and considering one of the sub - vedas ( upa - veda ) . therefore , its traditional holistic approach and principles observed to be have a potential in order to reduce the risk factors of chronic diseases in the community . it was already evident that cam had been widely used in africa ( 80.0% ) , australia ( 49.0% ) , indonesia ( 40.0% ) , france ( 75.0% ) , and even in united states ( 29.0 - 42.0% ) . in india as well , people are using the cam services through 2860 large hospitals across the country . cam has been integrated into primary health - care of the country under the national rural health mission . pattern of reported diseases at the cam facilities has not been shared with the modern medicine in india despite the provision of care to a significant group of the population . therefore , the present study was undertaken to describe the clinical profile of patients with chronic diseases at a tertiary care ayurvedic hospital in north india . the study was a part of assignment for the fellows appeared for 3 months course on non - communicable disease and health promotion at department of community medicine , dr . total 4 fellows appeared for the course and a 1 month field based assignment was allotted . during the course period fellows were assigned a mentor and a topic for study . present study was hospital based record based descriptive study carried out at department of kayachikitsa , rajiv gandhi post graduate ayurvedic college , kangra , himachal pradesh . as per census 2011 , the provisional population of the himachal pradesh was 68 56 509 ( 49.3% females ) and 15 07 223 ( 50.3% females ) of kangra district of the state . complied monthly data were accessed from the outpatient department ( opd ) records section from january 2011 to october 2011 . a single person collected the number of patients with an available diagnosis for every month . in addition , 30 case records ( patient file ) of admitted patients of hypertension and diabetes mellitus were selected randomly - using a random number table - from the inpatient department ( ipd ) . a total 30 case records were decided to study treatment practice at a tertiary care center . data were entered and analyzed by epiinfo 3.2 statistical software . before the data collection , ethical clearance form institution ethics committee and consent from studied health institutions was obtained . significantly ( p = 0.00 ) more males ( 51.1% ) reported at opd more than males . total 10,276 ( 15.8% ) children up to 5 years of age also sought treatment . almost half ( 53.1% ) of the patients were remained unclassified and kept under other category . commonly reported morbidities were related to respiratory ( 10.5% ) , neuromuscular ( 9.5% ) , digestive ( 9.2% ) , circulatory ( 9.1% ) , and eye ( 8.4% ) system . monthly trend of reported patients in the opd of kayachikitsa at rajiv gandhi post graduate ayurvedic college , kangra , himachal pradesh , 2011 ipd records of 30 hypertensive patients at ayurvedic tertiary hospital showed that the chest pain was the chief complaint ( 63.3% ) with mild to moderate intensity ( 70.0% ) and of radiating ( 56.7% ) in nature . it was observed that more than half ( 56.7% ) of the patients sought treatment from private practitioners before the admission [ table 2 ] . admitted patients had a history of hypertension for average 9 months and were on regular treatment for 21.3% of disease duration . diabetes mellitus had long standing history of average 145 month among the admitted patients , and since diagnosis ( 90.3% ) were on regular treatment . majority of patients with hypertension were being managed with aswaganda ( 66.0% ) and tagar powder ( 56.6% ) . symptomatic and management profile of patients in ipd of kayachikitsa at rajiv gandhi post graduate ayurvedic college , kangra , himachal pradesh , 2011 elevated blood pressure along with overweight and high cholesterol is a known risk factor for many chronic diseases . the chronic diseases are contributing about 46.0% of the all - cause mortality in india . evidence from india reported prevalence of chronic diseases up to 30.0% in the general population . it had been found to be prevalent in urban ( 36.4% ) along with slum ( 25.4% ) and rural ( 24.0% ) area . the present study at rural tertiary care health institute of ayurveda , showed that most of the patients were suffering from disorders related to respiratory , neuromuscular , and circulatory system , mostly suggestive of chronic diseases . based upon the grouped opd data it was observed to be difficult to further ascertain the causes of morbidity such as heart attack , brain attack , diabetes mellitus etc . , majority of patients were even not classified at all despite the complete diagnosis at the opd . it hindered the information that could ascertain that whether these patients were affected with the exact cause of chronic or acute disease . in the studied state , a total 27 ayurvedic hospitals with 580 beds , 1105 dispensaries and 7326 registered practitioners exist at the time of study . in the country , a total of 254 graduate and 64 post graduate ayurvedic colleges were producing 1197 under and 1110 post graduates every year . in addition , a total of 2458 hospitals with 44820 beds , 15353 dispensaries , 478750 registered practitioners . in ayurveda , dietary factors like unhealthy diet considered as a known risk factor for chronic diseases like consumption of high glycemic index , junk and salted food . however , food was being considered as medicine in ayurveda like barley / turmeric for obesity , kulatha for rheumatoid arthritis , ashwagandha as a general tonic . ayurveda had been playing a vial - role in the provision of treatment for common morbidities in india . ayurvedic products like jams , jellys , juices , biscuits , ice - cream , candies , chocolates , granules , flour , tea etc . , were recommended as healthy food and to be promoted to the crowd out the junk food in the existing market . therefore , with a vast human resource and the available ayurvedic treatment potential in the country , the rising trend of chronic diseases due to non - healthy life - style and dietary behavior could be managed successfully . furthermore , the actual burden and types of chronic disease in cam discipline need to be studied and shared . in this respect , standard method of case reporting modified as per the discipline to integrated disease surveillance project ( idsp ) at all levels and types of cam health facilities would certainly help to capture exact disease burden . as , government of india had already declared the mainstreaming of cam in modern health - care delivery system as it had recommended by appointment of at least one cam specialist at the community health center . ayurveda was being the oldest and the largest cam discipline in india , so , the integration of information from the ayurvedic health institution through idsp would be required to help for formulation of healthy public policy .

the american society of clinical oncology guidelines recommend sentinel lymph node biopsy ( slnb ) for all patients with melanoma tumors of intermediate thickness ( between 1 and 4 mm ) . in case of patients with thick melanoma tumors ( > 4 mm ) , slnb may be recommended as well , for staging purposes and to facilitate regional disease control . we report a case of an 82-year - old man , undergone excision of a cutaneous melanoma of the right thigh , which shows some limitation of slnb in thick melanoma . lymphoscintigraphy , performed as single - photon emission computed tomography / computed tomography ( spect / ct ) , failed to identify the real sentinel lymph node , as tracer uptake was seen in a right inguinal node . due to the presence on ct co - registered images of another suspicious node ( with no radiopharmaceutical uptake ) in the crural region , and considering the high - risk pathologic features of the removed primary lesion , a 18f - fluorodeoxyglucose positron emission tomography / ct ( 18f - fdg pet / ct ) staging scan was planned . pet / ct showed high metabolic activity in the suspected crural lymphadenopathy . histopathology demonstrated massive invasion of the crural ( sentinel ) node and no metastatic cells in the inguinal node . this report highlights both the higher accuracy of lymphoscintigraphy , when performed as spect / ct and the potential utility of 18f - fdg pet / ct in regional staging . since a very long time , a significant number of patients have been seeking treatment at complementary and alternative medicine health facilities , but the disease burden at these facilities has never been assessed and documented . present cross - sectional study was carried out at ayurvedic tertiary care hospital to document and to assess the rationale of disease reporting at ayurvedic institutions of the northern state of india from january 2011 to october 2011 . almost half of the patients morbidities were not classified at all into any of the disease categories . the common reported morbidities at study hospital were : respiratory ( 10.5% ) , neuromuscular ( 9.5% ) , digestive ( 9.2% ) and circulatory ( 9.1% ) disorders . as the majority of diseases were unclassified , so mainstreaming of the effective disease surveillance would be required to understand the morbidity pattern and successful treatment practices at health facilities .

proinflammatory cytokines ( il-1 , il-6 , and tnf- ) may significantly influence the development of obesity and concomitant disorders such as type 2 diabetes , arterial hypertension and metabolic disorders . it is suggested , that il-6 may affect the increase of free fatty acids level . tnf- is secreted mainly by macrophages and lymphocytes in response to cell damage caused by infection or malignant transformation . however , it can be also secreted by many other type of cells and tissues , e.g. , adipocytes . tnf- , similarly to il-6 , is a proinflammatory cytokine , characterized by a broad spectrum of functions which also include cytotoxic and cytostatic effects against cancer cells . it is able to inhibit lipoprotein lipase ( lpl ) at both mrna and protein levels . it also inhibits the expression of two major adipose tissue differentiation regulators : ccaat transcription factor that increases binding of alpha proteins ( cebp- ) and nuclear receptor ppar-2 . in consequence , changes in the expression of adipose tissue proteins , such as lpl , ap2 , fatty acid synthase , acetyl - coa carboxylase , glycerol 3-phosphate dehydrogenase ( gpdh ) , glucose receptor glut-4 , and others are observed . the aim of the present study was to assess the relation between il-6 and tnf- levels in serum of obese and normal weight subjects and to relate it to the crp concentration in obese subjects . the study was performed in accordance with the declaration of helsinki for human research and study protocol was approved by the ethics committee of warsaw medical university in warsaw , poland . kg / m , including 54 males aged 31 - 77 ( mean 53 1se years ) and 26 females aged 39 - 75 ( mean 61 2 years ) . a control group included 53 healthy subjects with normal weight , bmi < 25 ; 24 males aged 22 - 72 ( mean 39 2.3 years ) and 29 females aged 23 - 78 ( mean 47 2.4 years ) . in the studied population the tests were performed in a disease - free period , at least 3 weeks after any last infection , antibiotic treatment , and at least 3 months after any severe disease . blood samples were collected using edta as anticoagulant . to determine blood plasma concentration of il-6 and tnf , ' quantikine human il-6 ' and ' quantikine human tnf ' ( r&d systems , mn , usa ) elisa assay kits , respectively , were used . the measurements were taken using a microplate reader stat fax 2100 ( awareness technology , usa ) . crp plasma concentration was determined with fixed - point immuno - rate method ( ortho - clinical - diagnostics ) . results were analyzed for the whole population as well as in relation to gender , and compared with the control group . mean cytokine levels in different groups were compared with the mann - whitney u test . the mean il-6 level was generally lower in the control group in comparison with the population of patients population . however , a statistically significant difference in il-6 was found only between obese and normal weight women ( p = 0.001 ) , and not in males . the highest difference was found again with respect to the group of women ( p < 0.001 ) . in addition , il-6 level was higher in the obese patients with type 2 diabetes in comparison with the non - diabetic obese patients . here again , a statistically significant difference was observed only in the group of women ( p = 0.01 ) . the obese diabetic patients had a slightly higher tnf concentration than the obese non - diabetic patients ; the highest , albeit insignificant , difference being in the group of obese women . statistical analysis showed a positive correlation between il-6 and tnf concentrations ; significant values were achieved for the whole groups of overweight and obese patients ( p = 0.005 ) ( figure 1 ) . in whole populations , regardless of gender , a simultaneous elevation of both proinflammatory cytokines was observed . correlation between serum concentrations of il-6 and tnf ( r = 0.3 ; p = 0.005 ) . the crp level positively correlated with the il-6 concentration . here , the results for the whole overweight and obese group as well as in the women group were statistically significant . no statistically significant correlation between tnf and crp concentrations in overweight and obese patients was evident . correlation between il-6 concentration and crp level in the population of overweight and obese patients ( r = 0.3 ; p = 0.007 ) . the crp level significantly correlated with the obesity index expressed by bmi ; the correlation was observed in all analyzed subgroups ( figure 3 ) . correlation between bmi values and crp levels in the population of overweight and obese patients ( r = 0.4 ; p < 0.001 ) . it is widely suggested that overweight and obese subjects have elevated concentrations of inflammatory cytokines in serum . a possible mechanism of obesity - induced inflammatory response is based on the development of oxidative stress which , in consequence , leads to the inflammatory response induced by excessive intake of carbohydrates , mainly glucose . recent studies have shown that up to 30% of il-6 in the blood stream is secreted by adipose tissue , and the cytokine level positively correlates with body mass index ( bmi ) . intraperitoneal adipose tissue secretes three times more of il-6 than does subcutaneous adipose tissue . in the present study , moreover , a difference was observed in the population of obese , diabetic women in comparison with the women with no symptoms of diabetes . the obtained results suggest that gender is a major factor influencing the regulation of inflammatory factors ; possibly due to a specific hormonal balance or adipose tissue distribution in women . some authors consider both type 2 diabetes and insulin resistance as symptoms of an inflammatory response in the organism . the hypothesis can be confirmed by the increase in acute phase proteins , such as crp , amyloid a , alpha-1-acid glycoprotein , sialic acid , and cortisone in diabetic patients . the increase in il-6 , parallel to the elevated glucose and insulin concentrations , suggest a possible involvement of the cytokine in glucose metabolism , especially in adipocytes . likewise , in the present study we observed that cytokines positively correlated with the crp level , while crp , in turn , positively correlated with bmi in all analyzed subpopulations of obese patients . the increase in tnf expression in muscles and adipose tissue , through the tnf ability to phosphorylate serine residues in irs - i ( insulin receptor substrate 1 ) and a consequent decrease of insulin receptor tyrosine kinase activity , can lead to insulin resistance . the increase of tnf expression in the obese patients definitely demonstrated in the present study , can be a limiting factor for further enlargement of adipose tissue through the induction of insulin resistance of adipocytes . insulin resistance can influence lipoprotein lipase ( lpl ) activity and hence prevent excessive growth of adipose tissue . a high adipose tissue lpl to muscle lpl ratio observed in hyperinsulinemia leads to increased transport of lipids to the adipose tissue . so , rising insulin resistance will cause the reversal of the adipose tissue lpl to muscle lpl ratio , and in that way prevent further growth of adipose tissue . we suppose that tnf - induced insulin resistance , hyperinsulinemia , and higher lpl activity in adipocytes can cause body fat accumulation . on the other hand , the increase in body fat may lead to increased secretion of tnf that , in consequence , can cause insulin resistance . it is hard to state unambiguously whether the body fat content is a cause or a consequence of the observed differences in tnf concentration . however , it seems that the development type 2 diabetes depends on a number of other factors being more potent that tnf , as we did not find any differences in the tnf levels between the obese diabetics and non - obese patients with no diabetes . the empa - reg outcome trial ( clinicaltrials.gov identifier , nct01131676 ) recently announced the effects of empagliflozin on the cardiovascular outcomes ( cvo ) and mortality in persons with type 2 diabetes mellitus ( t2 dm ) . in this seminal cvo trial ( cvot ) , 7020 patients with t2 dm with coexisting cardiovascular disease ( cvd ; myocardial infarction [ mi ] , stroke , or peripheral arterial disease ) were randomized to either 10 mg empagliflozin , 25 mg empagliflozin , or placebo , over and above standard of care . the primary composite outcome was a total of three endpoints ( death from cv causes , non - fatal mi , and non - fatal stroke ) , while the key secondary endpoint included a fourth endpoint the results of this study revealed a statistically significant reduction in the primary endpoint with empagliflozin use ( 10.5% in the empagliflozin group vs. 12.1% in the placebo group ; hazard ratio 0.86 ; relative risk reduction ( rrr ) 14% , 95% confidence interval 0.740.99 ; p = 0.04 ) . similarly , a rrr reduction is noted in death from cv causes ( rrr 38% ; 3.7% vs. 5.9% ) , hospitalization for heart failure ( rrr 35% ; 2.7% vs. 4.1% ) , and all - cause death ( rrr 32% ; 5.7% vs. 8.3% ) . however , the difference in rates of non - fatal mi and non - fatal stroke did not reach statistical significance . this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by the author . the university group diabetes program ( ugdp ) study , published nearly half a century ago , highlighted the negative effects of tolbutamide on cv health . the united kingdom prospective diabetes study ( ukpds ) , a large study with multiple arms , unearthed the beneficial impact of metformin in contrast to other drugs such as chlorpropamide in improving cvo in t2 dm . the stop - niddm trial , breaking new ground , suggested cvo improvement when acarbose was administered to persons with impaired glucose tolerance . similarly , the proactive trial on pioglitazone reported a reduction in the composite of all - cause mortality , non - fatal mi , and stroke in patients with t2 dm who have a high risk of macrovascular events . however , all of these studies were not designed as a cvot , and their primary aim was to assess glucose - lowering efficacy rather than improvement in cv health . advances in the understanding of the interlink between diabetes and cv disease ( cvd ) , an appreciation of the need to improve cvo in diabetes care , and the realization that effective glucose - lowering drugs could end up worsening cvo ( e.g. , muraglitazar ) , have led to a mandatory requirement for cvots in anti - diabetic drugs pending regulatory approval . it then became imperative for all newly developed glucose - lowering molecules to undergo cv safety analysis by performing a cvot . the first drug to report such an analysis , after institution of new regulatory requirements , was quick - release bromocriptine , which demonstrated cv safety in a trial which recruited 3070 subjects for a mean follow up of 52 weeks . since then , various data on dipeptidyl - peptidase 4 inhibitors [ 810 ] and insulins have been published from large - scale cvots which have suggested their cv safety . while these cvots follow similar trial designs and protocols , subtle variations are present [ 811 ] . differences in inclusion / exclusion criteria , the duration of follow up , and the choice of primary / secondary endpoints can be easily discerned . though debate around the validity of various trial designs is never ending , there is broad consensus that modern cvots are well designed and have good reliability . all recent cvots have been designed to demonstrate cv safety ( non - inferiority ) , rather than superiority , as requested by regulators . over the past few decades the standard of care for cv prevention has improved markedly across the globe . use of medical interventions such as aspirin , statins , angiotensin - converting - enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arbs ) , and beta blockers has helped to enhance cvo , especially in persons with t2 dm . though widespread use of these standard of care drugs allow easier demonstration of cv safety , their use makes it difficult for a new add - on therapy to demonstrate improvement in cvo . this is exactly what empagliflozin has achieved . in a high - risk cohort of subjects , with over 75% usage of statins , over 80% use of acei / arbs , and over 60% beta - blocker therapy , administration of empagliflozin was able to demonstrate added benefit in terms of cvo . this speaks for the strength of the molecule being studied , as well as the robust quality of trial methodology followed by the authors of the study . inclusion of a wide variety of high - risk subjects , from 42 different nationalities , enhances the global applicability of these results . the benefits of empagliflozin were noted early on in the empa - reg outcome trial , and continued throughout the study . this finding makes empagliflozin stand apart from other cvd preventive drugs , such as statins and ramipril , which demonstrated a cv benefit after a longer duration of therapy . whether this means that empagliflozin is an effective drug for secondary prevention of cvd , that is , the prevention of progression of cvd to death is open to debate . detractors point to the lack of statistical significance noted with respect to occurrence of non - fatal mi and stroke . however , this finding may be thought to reinforce the preventive power of empagliflozin : it may even be used in tertiary cvd prevention , as it helps improve outcomes in persons with t2 dm who experience an mi or stroke , ensuring that they survive the acute illness . thus , empagliflozin may be useful for tertiary prevention , rather than secondary prevention , of cvd . the number needed to treat , ( for all - cause mortality ) , which was 39 for empagliflozin over a period of 3 years , is much lower than that reported for other drugs , including ramipril and simvastatin [ 12 , 13 ] . the highly significant benefit of empagliflozin ( p = 0.002 ) in preventing hospitalization for heart failure raises hope for its use in this clinical situation . this finding is especially welcome considering the controversy related to the increased risk of heart failure hospitalization with other glucose - lowering therapies . empagliflozin , therefore , gets evidence - backed justification for use not only as a glucose - lowering therapy , but also raises hope for its potential as adjunctive therapy for cvd prevention ( table 1).table 1impact of empa - reg outcome study strengths robust methodology / clinical design multinational coverage ( 42 countries ) applicability across age , gender , cv phenotype clinically relevant inclusion / exclusion criteria clear - cut answers from straightforward statistical analysispositive answers empagliflozin is safe in high - risk cv patients with t2 dm empagliflozin improves cv outcomes in high - risk cv patients with t2 dm empagliflozin can be used safely in combination with other vascular - tropic drugs over an extended period of time empagliflozin can be used for prevention of cvd empagliflozin does not increase the risk of diabetic ketoacidosis or bone fracturesunanswered questions is the beneficial effect of empagliflozin a class effect , or a property unique to this molecule ? what are the mechanisms that account for the potential of empagliflozin to reduce cv outcomes ? is the beneficial cv effect of empagliflozin relevant to patients with t2 dm and low cv risk , and to patients with type 1 diabetes mellitus ? can empagliflozin be prescribed in acute coronary settings ? can empagliflozin be considered an adjuvant preventive therapy for cvd ? cv cardiovascular , cvd cardiovascular disease , t2 dm type 2 diabetes mellitus impact of empa - reg outcome study cv cardiovascular , cvd cardiovascular disease , t2 dm type 2 diabetes mellitus the empa - reg outcome results should be carefully interpreted to assess their impact on diabetes care and outcomes . while the findings for different subsets of patients need to be studied separately , the empa - reg outcome study also throws up a few interesting questions ( table 1 ) . will these beneficial results be relevant to persons with t2 dm and low cv risk , or to persons with type 1 diabetes mellitus ? whether the findings of empagliflozin can be extrapolated to other sodium - glucose co - transporter 2 inhibitors ( sglt2i ) are open to discussion . cvots are underway for both canagliflozin and dapagliflozin , and their results will decide whether the cv benefits of empagliflozin are a class effect or not . it is also uncertain if empagliflozin can be initiated , or continue to be used , in acute coronary settings , including unstable angina , mi , and heart failure . however , while translating cvot evidence to practice one should not lose sight of good clinical sense . this therapy should be accompanied by appropriate medication counseling and should not be prescribed to persons at risk of ketoacidosis or recurrent genital infections . while the empa - reg outcome study findings should be interpreted carefully to assess their impact on diabetes care and outcomes , the results go far beyond proving the superiority of empagliflozin in improving cvo . the seminal importance of these findings will ensure that this trial acts as a milestone in the fields of diabetology and cv medicine . the empa - reg outcome study raises the bar for future glucose - lowering drugs . such a development will help improve not only cvo , but also diabetes outcomes overall . the empa - reg outcome study may be just a small step for empagliflozin , but is a giant leap for diabetes care .

backgroundthe development of obesity and related disorders , e.g. , type ii diabetes ( t2d ) , hypertension , and metabolic disturbances is strongly related to increased levels in proinflammatory cytokines ( il-1 , il-6 , and tnf- ) . both il-6 and tnf- are secreted by adipocytes and their concentration correlates with the percentage and distribution of fat tissue in the body . both cytokines are the main factors responsible for the induction of acute phase proteins production ( e.g. , crp ) and to inflammatory state.objectiveto compare of tnf- and il-6 concentrations in serum from obese subjects with those in subjects with normal bmi and to analyze the relation between tnf- , il-6 , bmi and the inflammatory state as measured by the level of crp.material and methodsthe study included 80 obese subject ( 54 males and 26 females ) bmi > 25 kg / m2 . a control group consisted of 53 healthy subjects ( 24 males and 29 females ) with bmi < 25 kg / m2 . to determine the blood plasma concentration of il-6 and tnf , commercial elisa assay kits were used.resultsthe concentration of il-6 was lower in the control compared with the obese patients , but a significance difference concerned only female subjects ( p = 0.001 ) . tnf- concentration was significantly higher in all obese subjects ( p < 0.001 ) . a higher level of this cytokine was also found in patients with obesity suffering from t2 dm . a positive correlation was present between il-6 and tnf- concentrations . only did the il-6 level correlate with the concentration of crp in serum.conclusionsthe study confirmed that increased inflammatory cytokines lead to the persistence of inflammation in obese subjects . however , some other factors , such as gender , may contribute to the development of obesity - related inflammatory states . this article discusses the recently published empa - reg outcome trial , which assessed cardiovascular outcomes with empagliflozin therapy in persons with type 2 diabetes mellitus and coexisting cardiovascular disease . the article describes the background and challenges of modern cardiovascular outcome trials , points out the strengths of the empa - reg outcome study , and places the results in perspective . it highlights the significant impact that these results will have on cardiovascular preventive pharmacotherapy , and on future drug development in diabetes . at the same time , it reminds readers of the limitations of the results , and lists the questions raised by , or left unanswered by , the trial .

primary neuroendocrine carcinoma ( nec ) of the breast is a rare distinct clinicopathological entity , comprising 0.5 - 2% of breast carcinomas world - wide . in 2003 , primary nec of the breast was identified as a distinct entity by the world health organization ( who ) classification of tumors . the who classification defines primary nec of the breast as tumors that express 50% or more of ne markers there are no previous reports of f-18 fluorodeoxyglucose ( fdg ) positron emission tomography / computed tomography ( pet / ct ) in patients with primary nec of breast with liver and bone metastasis . here we describe a case of a 45-year - old female patient who presented with jaundice and was evaluated to have multiple liver lesions and biopsy from liver showed metastatic neuroendocrine tumor . her chromogranin level was 886 ng / ml ( normal < 78 ng / ml ) . she was referred for the whole body pet / ct for detection of the primary site . pet / ct showed an intense uptake in the soft- tissue necrotic lesion in the inner quadrant of the left breast [ figure 1a and b ] with the same charecteristic feature of multiple hypodense liver lesions [ figure 1c ] . furthermore uptake noted in a lytic lesion in the d4 vertebra [ figure 1d and e ] . patient underwent fine - needle aspiration of the breast lesion , which confirmed neuroendocrine origin [ figure 2a and b ] . a diagnosis of primary nec of the left breast with metastasis was made and she was treated with peptide receptor radionuclide therapy and is on follow - up now . whole body fluorodeoxyglucose - positron emission tomography / computed tomography ( pet / ct ) maximum intensity projection image ( a ) , axial fused pet / ct showed a intense uptake in the soft - tissue lesion in the left breast ( b ) , liver lesions ( c ) and bone lesion ( d ) , axial ct showing lytic bone lesion in d4 vertebra ( e ) immunohistochemistry staining showing positive for chromogranin ( a ) and synaptophysin ( b ) primary nec of the breast is extremely rare with the first reported case in 1983 . the most frequent reported age varies from 40 to 70 years , with a higher incidence in women greater than 60 years . as metastatic neuroendocrine tumors of the breast are more common than that of primary neuroendocrine tumors of the breast , it is , therefore , important to differentiate primary breast neuroendocrine tumor from metastatic disease to the breast because of the differences in treatment focus . primary nec of the breast can be diagnosed if the presence of a non - mammary primary site can be clinically ruled out or if an in situ component is histologically detected or both . however , findings of certain studies have revealed that ne - differentiated tumors of the breast present as dense round or irregular masses with spiculated or lobular margins on the mammogram . definitive diagnosis is made with core needle biopsy , allowing for the immunohistochemical evaluation of the specimen for the ne markers . although the use of pet for the evaluation of ne tumors has been limited , tumors with moderate or high proliferative activity can be identified by fdg pet . there are reports of fdg pet / ct in a case of neuroendocrine differentiated breast carcinoma with pleural metastases using indium-111 octreotide . there are case reports of synchronous metastases to the liver and pancreas from a primary nec of the breast . our case is the first demonstrates that 18f - fdg pet / ct provides the most significant additional information related to the accurate detection of primary nec of breast and bone metastasis and guiding treatment . malignant unilateral or bilateral ureteral obstruction may be secondary to direct tumor invasion , extrinsic compression , or encasement by metastatic retroperitoneal or pelvic lymph nodes . the selection of cancer patients for diversion should take into account factors such as tumor stage , prognosis of the primary cancer , likelihood of additional antineoplastic therapy , and quality of life . contemporary management options include external drainage via percutaneous nephrostomy ( pcn ) and internal drainage via the insertion of double - j stents . regular double - j stents used to relieve ureteral obstructions that are secondary to extrinsic causes , such as malignancies , have high rates of failure . pcn is commonly used as an alternative , either as a primary procedure or after the failure of transurethral procedures . however , pcn is more invasive than double - j stent insertion and may also have a greater incidence of accidental tube dislodgement . the invasiveness of the procedure and the high incidence of tube dislodgement may result in a reduction in patient quality of life . in addition , some patients are unwilling to accept a pcn tube because it requires an external collecting device . the limitations associated with conventional treatments for ureteral obstructions highlight the need for a novel treatment that can maintain ureteral patency while minimizing the deterioration of patient quality of life . several types of metallic stents have been used in the palliative treatment of malignant ureteral obstructions , but implantation of these stents has yielded results that are not uniform and the stents have been associated with various complications . a novel polytetrafluoroethylene ( ptfe ) membrane - covered metal mesh stent prevents obstruction from tissue ingrowth and reduces stent migration as a result of its unique structure . here we report our initial experience with a ptfe membrane - covered self - expandable metallic stent ( uventa stent ) for the palliative care of malignant ureteral obstruction . between october 2010 and november 2011 , 18 consecutive patients ( 5 men and 13 women ; mean age , 57 years ) underwent placement of uventa stents ( taewoong medical , seoul , korea ) for unilateral or bilateral malignant ureteral obstruction . the ureteral obstructions were caused by compression by a localized primary tumor in 1 ureter , remote metastatic disease or direct tumor infiltration in 6 ureters , and encasement by retroperitoneal lymphadenopathy in 13 ureters . six patients had uterine cervical cancer , 7 had colorectal cancer , 4 had stomach cancer , and 1 had retroperitoneal sarcoma ( table 1 ) . diagnostic imaging of obstructions was performed by using transabdominal ultrasound , computerized tomography ( ct ) , or intravenous urography ( ivu ) . a single urologist ( jwp ) candidates for uventa stent placement had pre - existing double - j stents that were either nonfunctional or caused excessive bladder irritation . the outer and inner meshes are made of nickel plus titanium alloy and there is a ptfe membrane between them ( fig . the inner shaft is pierced to the tip by the central lumen to allow the passage of a 0.035-inch guidewire . the stent is deployed by pulling the outer sheath back without moving the inner shaft . we used a uventa stent with a 7-mm nominal diameter that ranged in length from 6 to 12 cm . retrograde pyelography was performed to identify the obstructed ureteral segment shape , level , and length ( fig . stricture traversal was attempted by an appropriate combination of a 0.035-inch straight percutaneous transluminal angioplasty guidewire with hydrophilic coating or ptfe - coated guidewire and a 5.0 fr/70 cm ureteral catheter ( open - end flexi - tip , cook urological inc . , spencer , in , usa ) . gentle interventional maneuvers were necessary to prevent ureteral rupture and contrast extravasation , which might blur the fluoroscopic field and promote periureteral fibrosis . after successfully traversing the stricture , the guidewire was forwarded into the renal pelvis and exchanged for a rigid 0.035-inch amplatz super stiff guidewire ( boston scientific , miami , fl , usa ) to strengthen the ureteral course and secure the luminal passage during balloon dilation and stent placement ( figs . the stent was placed such that its upper and lower ends bypassed the stricture by 2 cm . when the obstruction site was in the distal ureter , the lower end of the stent was positioned intravesically , extending approximately 0.5 to 1 cm from the ureteral orifice ( fig . in long strictures requiring two or more uventa stents , the stents were placed sequentially , overlapping by 2 to 3 cm . balloon dilation was performed with a 6-mm balloon dilation catheter ( uromax ultra , boston scientific , galway , ireland ) . repeated high - pressure balloon dilation ( post - dilation pressure up to 20 atm ) was necessary in cases of resistant stricture and suboptimal stent expansion . after the intervention was completed , we planned follow - up visits with patients at 1 , 3 , 6 , and 12 months after stent implantation and yearly thereafter . urine culture , blood biochemistry tests , and ivu or ct were performed at the follow - up examinations to detect any recurrent strictures . all patients received specific instructions to return to our hospital in case of ipsilateral flank pain , abdominal pain , fever , dysuria , urgency , frequency , hematuria , or vomiting . additional uventa stent insertion was planned for any recurrent ureteral obstruction during follow - up . the outer and inner meshes are made of nickel plus titanium alloy and there is a ptfe membrane between them ( fig . the inner shaft is pierced to the tip by the central lumen to allow the passage of a 0.035-inch guidewire . the stent is deployed by pulling the outer sheath back without moving the inner shaft . we used a uventa stent with a 7-mm nominal diameter that ranged in length from 6 to 12 cm . retrograde pyelography was performed to identify the obstructed ureteral segment shape , level , and length ( fig . stricture traversal was attempted by an appropriate combination of a 0.035-inch straight percutaneous transluminal angioplasty guidewire with hydrophilic coating or ptfe - coated guidewire and a 5.0 fr/70 cm ureteral catheter ( open - end flexi - tip , cook urological inc . gentle interventional maneuvers were necessary to prevent ureteral rupture and contrast extravasation , which might blur the fluoroscopic field and promote periureteral fibrosis . after successfully traversing the stricture , the guidewire was forwarded into the renal pelvis and exchanged for a rigid 0.035-inch amplatz super stiff guidewire ( boston scientific , miami , fl , usa ) to strengthen the ureteral course and secure the luminal passage during balloon dilation and stent placement ( figs . the stent was placed such that its upper and lower ends bypassed the stricture by 2 cm . when the obstruction site was in the distal ureter , the lower end of the stent was positioned intravesically , extending approximately 0.5 to 1 cm from the ureteral orifice ( fig . in long strictures requiring two or more uventa stents , the stents were placed sequentially , overlapping by 2 to 3 cm . balloon dilation was performed with a 6-mm balloon dilation catheter ( uromax ultra , boston scientific , galway , ireland ) . repeated high - pressure balloon dilation ( post - dilation pressure up to 20 atm ) was necessary in cases of resistant stricture and suboptimal stent expansion . after the intervention was completed , we planned follow - up visits with patients at 1 , 3 , 6 , and 12 months after stent implantation and yearly thereafter . urine culture , blood biochemistry tests , and ivu or ct were performed at the follow - up examinations to detect any recurrent strictures . all patients received specific instructions to return to our hospital in case of ipsilateral flank pain , abdominal pain , fever , dysuria , urgency , frequency , hematuria , or vomiting . additional uventa stent insertion was planned for any recurrent ureteral obstruction during follow - up . two patients had bilateral ureteral obstructions , resulting in a total of 20 ureters that were managed by uventa implantation . mean ureteral obstruction length was 10.6 cm ( range , 2 to 20 cm ) . the first cystoscopic attempt failed in one patient because direct tumor invasion into the uretero - vesical junction made it impossible to find a ureteral orifice . the patient then underwent an anterograde double - j stent insertion , and we used the double - j stent as a guide for cystoscopic retrograde uventa insertion . we performed simultaneous balloon dilation in 12 ureters ( 60% ) because there was insufficient expansion of the uventa stent ( < 50% of diameter ) after deployment . the procedure time varied with the length and severity of obstruction , but ranged between 30 and 60 minutes . abnormally elevated serum creatinine decreased to normal levels ( table 1 ) and hydronephrosis gradually resolved by 4 weeks after uventa insertion ( fig . two patients experienced mild lower abdominal pain and discomfort for a few days after uventa deployment , probably as a result of the expanding force of the endoprostheses . two patients reported irritative bladder symptoms that may have been due to the protrusion of the distal end of the uventa stent into the bladder . mean follow - up time was 7.3 months ( range , 3 to 15 months ) . follow - up imaging studies ( ct or ivu ) indicated no stent obstruction by hyperplastic reaction or tumor ingrowth and the uventa stents maintained their shape . there were no instances of stone formation , encrustation , or migration of the uventa stents . the first report of double - j stents as a treatment for ureteral obstruction was published in 1978 . subsequently , these stents have been widely used for patients with extrinsic ureteral obstructions due to malignancy . however , the use of double - j stents in malignant ureteral obstructions has a high rate of failure . metallic stents have been applied in cardiac , gastroenterological , hepatobiliary , and vascular systems . several authors have proposed metal stents to ameliorate obstructive urinary tract pathology , including benign prostatic hyperplasia , urethral stenosis , ureteroileal anastomotic stricture , benign and malignant ureteral obstruction , and even kidney transplantation ureteral stenosis . self - expandable metallic stents have been used to treat malignant ureteral obstruction with acceptable results , but they can be obstructed by hyperplastic reactions or tumor ingrowth through the stent struts . in a description of their 10 years of experience with metal mesh stents , liatsikos et al . reported that the most common complications that jeopardized ureteral patency were hyperplastic reactions and tumor ingrowth . these complications developed in 45 of 119 ureters . the externally coated ureteral metallic stents and thermo - expandable metallic ureteral stents that were introduced to limit the ingrowth of hyperplastic tissue through stent struts frequently migrate , with migration occurring in 17.5 to 81.2% of these stents . this stent migration may be attributed to the lack of an appropriate anchor to the ureteral wall for the prostheses and propulsion by anterograde peristalsis . the unique structure of the uventa stents used in the present study prevents not only tissue ingrowth but also stent migration . the inner mesh and ptfe membrane prevent tissue ingrowth into the stent and the outer mesh induces ingrowth of tissue that acts as an anchor between the stent and the ureteral wall , thereby preventing migration . in a study comparing ptfe - covered metallic stents with uncovered metallic stents in canine ureters , chung et al . observed tissue proliferation on the outside of the covered stents but not within the lumen . we found no evidence of migration or obstruction of the stents in any of the follow - up imaging studies . an important complication associated with long - standing double - j stents is encrustation and stone formation with subsequent urinary tract infection and obstruction . stent encrustation rates were reported to be 12 to 27% in series of studies with memokath 051 thermoexpandable metallic stents . the encrustation rates of covered metal mesh stents are not yet well known . because metal stents are usually implanted in situ rather than being regularly changed like double - j stents , encrustation is a serious concern . in the present study , although we did not search for stone formation or encrustation by use of endoscopy , there was no stent obstruction from stones or visible encrustation in radiologic studies . some of the patients in this study experienced minor postoperative complications , such as gross hematuria , abdominal pain , or irritative urinary symptoms . however , there were no serious complications of grade iii or higher by clavien classification . variable patency rates ( 51.2 to 100% ) are reported for various types of metallic ureteral stents used in the treatment of ureteral obstruction irrespective of cause . in our study , the uventa stents were not obstructed during follow - up , so that the overall patency rate was 100% . however , de novo ureteral obstruction by tumor progression occurred in 4 ureters : 3 adjacent to the lower end , and 1 adjacent to the upper end of the implanted uventa stent . the other 2 patients were in the terminal stage of cancer , and no additional procedure was performed . however , a percutaneous approach requires the assistance of an interventional radiologist and an additional invasive pcn procedure . many urologists are already familiar with the cystoscopic retrograde double - j stent insertion , and the procedure for insertion of uventa stents is virtually the same . we performed all procedures by using a retrograde approach and easily implanted the stents in all but one patient . if the obstruction is in the lower ureter and uretero - vesical junction and may be due to invading cancer , it is helpful to use cystoscopy to identify an ipsilateral ureteral orifice before the procedure . when such an orifice can not be identified , preoperative pcn and anterograde double - j stent insertion is necessary for successful uventa stent implantation . when the obstruction site is in the upper or midureter , it is better to conduct balloon dilatation after uventa insertion because the proximal and the distal ends of the stricture site can not be accurately identified after balloon dilation is complete . when the obstruction is in the distal ureter , we advise positioning the lower end of the stent intravesically , extending approximately 0.5 to 1 cm from the ureteral orifice . this will reduce the risk of possible uretero - vesical junction obstruction by later tumor invasion . the recently developed uventa stent prevents tumor ingrowth through the mesh and is resistant to stent - related complications . therefore , it obviates the need for regular stent changes and thus offers significant benefits for patients with limited life expectancy . although we did not find any serious stent - related complications in the short term , a longer follow - up period is required to confirm the safety and efficacy of uventa stent insertion . additionally , we did not evaluate stent - related symptoms and quality of life changes by use of validated questionnaires . it is essential to determine whether there are any benefits of uventa stents over double - j stents by conducting a comparative study . we are planning a prospective comparative study of this type to better evaluate the potential of uventa stents for the treatment of ureteral obstruction . ptfe membrane - covered self - expandable metallic stents can relieve malignant ureteral obstructions safely and easily . long - term follow - up is required to assess the role of these stents in the treatment of malignant ureteral obstructions .

cases of primary neuroendocrine carcinoma ( nec ) of the breast have been reported , though rare . we report the case of a 45-year - old woman presented with jaundice and evaluated to have liver metastasis from neuroendocrine origin . she underwent whole body positron emission tomography / computed tomography , which showed left breast lesion and bone metastasis . fine - needle aspiration ( fna ) of breast revealed a nec . a diagnosis of a primary nec of the breast was rendered with hepatic and bone metastasis . she was treated with peptide receptor radionuclide therapy and is on follow - up . purposewe assessed the efficacy and safety of insertion of a polytetrafluoroethylene membrane - covered self - expandable metallic stent ( uventa stent ) for palliation of malignant ureteral obstruction on the basis of our early results.materials and methodseighteen patients underwent uventa stent insertion for extrinsic malignant ureteral obstructions of 20 ureters . the uventa stents were deployed retrogradely under cystoscopy and fluoroscopy . candidates for the procedure had preexisting double - j stents that were nonfunctional or caused excessive bladder irritation . we recorded the success and patency rate in addition to any complications associated with the procedure.resultsthe mean length of obstruction was 10.6 cm ( range , 2 to 20 cm ) . two ureters were obstructed in the upper ureter , 9 in the lower ureter , and 9 in multiple levels of ureter . simultaneous balloon dilation was performed in 12 ureters . uventa stents were successfully inserted in all patients . no obstruction of the uventa stents occurred during the mean follow - up period of 7.3 months ( patency rate 100% ) , but de novo ureteral obstruction developed in 4 ureters . there were no instances of stone formation , hyperplastic reaction , encrustation , or migration . abnormally elevated serum creatinine decreased to normal levels and hydronephrosis gradually resolved during the 4 weeks after uventa insertion . no significant complications developed except for transient and self - limiting hematuria and mild lower abdominal pain.conclusionsuventa stents may relieve malignant ureteral obstruction safely and easily . long - term follow - up is necessary to assess the role of this stent in the treatment of malignant ureteral obstruction .

typically , using standard assessment criteria , approximately 50 to 100 channel targets are detected in chondrocytes . not surprisingly the dataset from each study are rather different to each other , but there is a common set of 7 probes that are detected in all 10 of these studies . it is important to note that this analysis does not select particular ion channel genes to study , but reveals all the genes commonly expressed in the above microarray datasets ( note that there are over 300 ion channel gene probes encoded on each of the cited affymetrix chips ; accessory proteins such as the channel tetramerization domain proteins are excluded ) . with such a large number of ion channel transcripts on the affymetrix chips one would expect the random co - detection of transcripts across datasets . to quantify this we used the following statistical analysis . the probability of more than n transcripts being co - localised in d datasets is given by : where p is the probability of a given gene appearing in a datasets , and there are a total of g genes on each array . this gives a p - value for 7 ( or more ) transcripts appearing in all 10 datasets by chance as p < 1e-14 . it should be noted that these microarray datasets were derived from different species ( 3 rat , 3 mouse , 3 human and 1 bovine ) and there are potential differences in chondrocyte isolation protocols . constraining analysis to just rodent ( 6 datasets ) returns a set of 23 commonly expressed ion channel genes ( table 2 ) . figure 1 quantitatively illustrates both the overlap of genes commonly expressed between species ( fig . 1a ) and the overlap between each of the transcripts from human , mouse and rat ( fig . it is evident that far more transcripts were detected in all 3 of the mouse datasets than in all 3 of the human datasets . this could be for three reasons ; firstly , it is possible that the sensitivity of the mouse chips is greater , but we have seen no specific evidence for this . secondly , each of the protocols requires manual dissection and separation of chondrocytes from the subchondral bone and adnexa . samples are inherently contaminated with non - chondrocyte tissue . in an electrophysiological or immunohistochemical study such contamination would be relatively easy to detect , but in a biochemical protocol , where harvested tissue is macerated and then processed , it could be missed . thirdly , it is conceivable that there are genuine phenotypic differences between chondrocytes in mice and other animals . these represent transcripts present in each of the 3 datasets for each of the human , mouse , and rat datasets ( i.e. , includes 9 datasets total ) . the human studies used tissue harvested from either adolescents receiving limb length correction surgery ( e - geod-1277 ) , adults receiving acl surgery ( e - geod-16464 ) , or post mortem ( e - geod-10024 ) . samples were taken from knee ( e - geod-16464 ) , distal femur ( e - geod-1277 ) , or shoulder ( e - geod-10024 ) and focussed on articular ( e - geod-10024 ) , mixed ( e - geod-16464 ) , or growth plate ( e - geod-1277 ) chondrocytes . chip ids ; e - geod-10024 used hg - u133a and e - geod-16464 used the slightly newer hg - u133a_plus_2 , but e - geod-1277 used the u95av2 genechip . all 3 human studies used expanded chondrocytes , but e - geod-10024 and e - geod-16464 re - constituted those into 3d cultures . extraction enzymes were collagenase p ( e - geod-10024 ) , clostridial collagenase and deoxyribonuclease i ( e - geod-16464 ) and trypsin ( e - geod-1277 ) . rodent studies suffer from inherent difficulties in extraction of tissue since cartilage is thinner than larger animals . tissue used from the microarray studies analysed in this letter came from a variety of joints from immature mice and are likely to include mixed chondrocyte phenotypes . where stated explicitly , chondrocytes were expanded in monolayer cultures following collagenase based isolation ( e - geod-8052 and e - geod-7683 ) . all 3 studies ( e - geod-10556 , e - geod-18052 and e - geod-7683 ) used the same affymetrix mouse430_2 chips . the rat femoral head ( e - geod-6119 , e - geod-14402 ) or knee ( e - geod-8077 ) tissue was harvested from a range of ages from one day old neonates ( from which only the outer two - thirds of cartilage was used to select for articular chondrocytes , e - geod-14402 ) to several month old rats ( 300 - 320 g , e - geod-8077 ) . strain was either wistar ( e - geod-6119 ) , sprague - dawley ( e - geod-8077 ) or not stated . e - geod-6119 and e - geod-14402 both used monolayer expanded chondrocytes following collagenase ii based isolation . e - geod-6119 also included pronase , but e - geod-8077 used direct rna extraction from macerated tissue . one bovine dataset derived from chondrocytes 3d cultured from carpal bones of 3 to 6 mo old calves was also analyzed ( e - geod-18394 , affymetrix bovine chip , annotated with version since there was only 1 bovine chondrocyte dataset on ebi ( albeit including a number of replicates ) this is not included in the venn diagrams . note that each of the 3 species sets in ( a ) is equivalent to the commonly expressed regions of the venn diagrams in ( b , c , and d ) . the remainder of this letter focuses on whether the microarray data provides useful clues as to which channels are expressed in chondrocytes , and whether , therefore , initial processing of microarray data will improve the rate of channel detection in chondrocytes , or potentially other tissues . the chloride channel superfamily is huge and includes the large clc family , cftr protein , calcium - activated , volume - activated , p64 related chloride channels ( clns ) , and intracellular chloride channels ( clic ) . in many systems chloride channels have been less well studied than cation channels , although they were some of the earliest ion channels identified in chondrocytes . analysis of the current datasets reveals that both p64-related ( clic1 , clic4 ) and clns1a ion channels were detected in all 10 microarray experiments . the function of the corresponding channels are unknown , complex , controversial , or a combination of all these . clic1 , clic4 , and other members of the clic family of proteins appear to be legitimate anion channels . they are often referred to as p64-related simply because their earliest characterization appeared to be of a 64kda protein . they appear to be of relatively low conductance for a chloride channel ( 8 - 40ps , depending on experimental conditions ) . whilst , as their name implies , these channels can localize to intracellular compartments , in some cell types they also appear to be in the plasma membrane and could serve a role in secretion . one possibility is that the channel shuttles to and from the membrane in a cell cycle dependent way . the pcln channel ( sometimes referred to as p - glycoprotein , picln ) , also detected in 10 out of 10 datasets , was first identified by paulmichl et al as a putative rather ubiquitous volume - sensitive chloride channel . more recently , this volume regulator role has been refuted and roles in gene - regulation and development have been proposed . the controversy surrounding the nature of this channel is discussed in detail by strange and furst . the particular issue is that it can not be clearly determined whether this is a volume - sensitive channel , another type of channel , or is a regulator of a channel endogenous in the various expression systems in which it has been studied . even the fundamental property of ion selectivity is controversial , since recent studies have shown a rather higher permeability of pcln to cations than would be expected for a chloride channel . it is possible that one or other of these channels does constitute the chloride channel identified by tsuga et al in chondrocytes , but it is unlikely since that channel is more typical of a classical maxi - type chloride channel in electrophysiological and pharmacological terms . the associated channels are thought of , generally , as mitochondrial ion channels , found in the outer membrane of this organelle . the proteins have also been detected in the plasma membrane , where they exhibit voltage - gated anion channel activity ( based on data in the ncbinr and uniprot databases ) . vdac channels are also implicated in apoptosis and , as such , they will be of profound importance to all cells in which they are expressed . it has also now been suggested by a number of authors that some vdac protein expression is also of plasma membrane ion channels . this would be very much in line with the original chondrocyte chloride channel work of tsuga and sugimoto . the channel identified in these studies was the maxi - chloride channel , which is remarkably similar to the maxi - cl / vdac channel . in our own unpublished work , we see clear expression of a large conductance , niflumic acid and sits - sensitive chloride channel which appears likely to be maxi - chloride . it is no surprise that kcnma1 ( bk ) has been detected in all of the 10 datasets analyzed here . currents either broadly , or specifically , identified as being through bk channels have been described in a number of papers . in our own work we have shown not only that bk currents are present by electrophysiology , but also that the kcnma1 was detectable by immunohistochemical methods . interestingly , whilst the bk function modifying -subunit kcnmb1 was detected in our own immunohistochemical studies it was not detected in any of the 10 microarray studies discussed here . there was detection of kcnmb2 and kcnb4 in 2 and 3 out of the 10 studies respectively . activation of bk results in such large currents that it is likely to be involved with regulation of intracellular osmolarity and volume . early work did not show whether this activation was direct or indirect via an increase of intracellular calcium ions , but our more recent studies show rather convincingly that trpv4 is activated by stretch and that this results in the opening of a potassium channel . the detection of the sodium 1-subunit scn1b in all 10 studies is interesting , since the -subunits were undetectable in most studies . this subunit has been shown , in neurons to convey subtle changes to expression patterns and functional properties of voltage - gated sodium channels . there has been 1 electrophysiological study , which identified voltage - gated sodium channels in chondrocytes . this work has not been followed up on and , since chondrocytes do not fire action potentials , it is difficult to see what the role of a voltage - gated sodium channel might be . for completeness : scn7a ( nav2.1 ) , a somewhat atypical na channel was detected in 4/10 experiments , and the classical sodium channel scn2a1 in 3/10 , and the transient type sodium channel scn11a and scn10a in 2/10 datasets . this list includes trpc1 , of the canonical trp , and trpv4 , a vanilloid channel , both of which were identified in 9/10 microarray studies . in traditional protein and pharmacological studies , however , three trp channels have been identified in chondrocytes trpv4 , trpv5 , and trpv6 but not trpc1 . trpv5 appears important for setting the membrane potential , crucial to maintenance of cell volume in chondrocytes . trpv4 , however , appears to be critical for allowing entry of ca and activation of bk channels during imposed volume increase , and thus the process of regulatory volume decrease in epithelial cells . also , however , we find that 2 transient receptor potential channels are commonly detected . to use traditional methods to identify changes in channel expression between populations of cells from different tissues is probably not feasible unless high throughput automated ion channel recording equipment can be adapted for this purpose . however , observation of changes in cellular properties and mathematical models may provide clues as to which channels have changed in oa . an alternative approach would be to use microarray comparisons between normal and oa tissue , detect changes in channel mrna abundance and then follow - up with functional or histochemical experiments . generally , differential expression patterns from microarray studies are used to identify changed pathways , however , this process could miss changes which take place in individual ion channels not associated with an established pathway . the next microarray study considered in this review specifically compared transcript abundance from chondrocytes in human normal and oa cartilage . the authors focused on ( and verified ) changes in many cartilage phenotypic markers , but did not specifically consider changes in other proteins such as ion channels . probing this list for ion channels and porins significantly changed ( greater than 2-fold change ) produces the data in table 4 . the data clearly show an approximately 3-fold change in enac , tmem16a and bk ( kcnma1 ) transcript abundance and a 38-fold change in the aquaporin aqp1 channel transcript abundance . interestingly each of these channels are important for chondrocyte cell volume control . from this , by way of proof of principle we verified bk channel changes with immunohistochemistry and aquaporin channel changes with a functional assay . negative is fold decrease in abundance ratio , positive is fold increase in abundance ratio . note that whilst there were probes for trpv4 present on the chip , the huge variability between abundance scores between samples for this particular probe set make it unlikely that any change would be detectable , even if there was one . tissue was taken from stifle joints of horses with and without oa ( see fig . we investigated the expression of both - and -subunits of bk ( only kcnma1 was included in karlsson 2010 , there were no probe sets for kcnmb1 on the chip ) . semi - quantitative analysis of protein expression density shows that both bk subunits were significantly increased in oa , in the middle zone ( fig . immunohistochemical identification of kcnma1 ( bk -subunit ) and kcnb1 ( bk -subunit ) in sections of healthy and oa equine cartilage . macroscopically normal articular cartilage samples were obtained from weight - bearing regions of the metacarpophalangeal joints of horses of mixed breed , age , and sex . animals were euthanized for non - research purposes having been stunned before slaughter for meat in accordance with welfare of animals ( slaughter or killing ) regulations 1995 . sections of normal ( n=6 ) and oa ( n=3 ) equine cartilage were probed for channel expression by immunohistochemistry essentially as previously described . sections were incubated overnight at 4c with rabbit polyclonal antibodies to the kcnma1 and kcnb1 . antibody dilutions used ranged from 1:200 to 1:1500 in tris - buffered saline containing 1% bovine serum albumin . slides were incubated with horseradish peroxidase - labelled polymer conjugated to affinity - purified goat anti - rabbit immunoglobulins . photomicrographs of immunostained tissue sections captured using nikon digital sight ds-5 m camera driven by nikon eclipsenet image capture software ( nikon ) . positive staining is indicated by brown staining and particular evident at middle / superficial zones . the largest increase in expression density ( from data such as that illustrated in fig . 2 ) is in the middle zone , for both kcnma1 ( c ) and kcnmb1 ( d ) ( - and - subunit respectively ) . cells are challenged with a hypotonic solution , causing them to swell , and the rate of swell can be measured to determine the aquaporin expression . the bioinformatics showed a 38-fold increase in aqp1 transcript abundance in oa and we see a significant increase in functional aquaporin expression in tissue from oa joints ( fig . 3a ) , although the increase is much smaller than the change in aqp1 transcript abundance . current pharmacological tools do not allow categorical determination of aquaporin subtype , however , here , water permeability of chondrocytes was blocked by concentrations of bumetanide , tea , and hgcl2 consistent with that expected for aqp1 ( fig . water permeability can be calculated from the initial slope of the relative volume ( v / vo ) against time curve . where v is the volume at time ( a ) permeability is 303% ( p<0.05 , n=4 ) greater in chondrocytes from dogs with osteoarthritis ( oa ) . tea ( a blocker of aqp1 ) , bumetanide ( pic50 5.170.11 m , n=6 , bumex , a blocker of aqp1 and 4 ) , and mercuric chloride ( hgcl2 a non - specific aqp blocker reversed by 2-mercaptoethanol ) me ) are included to determine aqp type ( b and c ) . solution including 120 mm sucrose ( osmolarity 300mosm ) , then moved to an identical physiological saline without the sucrose . live cell imaging was achieved with a nikon diaphot microscope equipped with a sony icx098qb high sensitivity ccd . images were analysed offline with imagejvolume was calculated from the 2d surface area ( a ) of the cell disc by assuming the cell is approximately spherical as described previously , using the following equation : vol=43(a)3 ( equation 2 ) . except where stated , data are presented normalized for starting volume ( v0 ) as v / v0 , where v is the volume at time t. visual data were analyzed with imagej and anova performed with spss ( spss inc . ) . note that canine tissue was harvested from clinical waste tissue with local ethical approval , no dogs were harmed for the study . in summary , there appears to be considerable agreement between transcriptomic studies and physiological or immunohistochemical studies . it would seem that most channels common to all 10 datasets can be identified by these other techniques . there are examples , however , of proteins which have been identified in chondrocytes yet show up in few datasets . for example , the asic channel ( accn2 gene ) has been shown by immunohistochemistry and rt - pcr yet shows up in only one of the three rat datasets discussed here . therefore , combining these approaches should massively speed up the rate of discovery of ion channels in cell types which can be isolated in sufficient quantities to perform such studies . there are tissues , such as the brain , where cell types are too intermingled to allow identification of unique cell types , but for many tissues in the musculoskeletal system ( or cell lines ) , the combination of transcriptomics and protein studies seems ideal . with regard to oa , this strategy has allowed us to very rapidly pinpoint some phenotypic changes in the expression of two important channels in oa : an aquaporin and the bk channel . further protein and functional experiments will be necessary to establish whether the other kca channels are also altered , and in particular whether these changes contribute to or result from progression of oa . chronic obstructive pulmonary disease ( copd ) is characterized by progressive and partially reversible airflow limitation , and it is among the leading causes of mortality worldwide.1 copd is a multicomponent disease , and patients present a range of comorbidities that have an impact on prognosis and may increase the risk of mortality.2 the effects of copd on respiratory and physical function have been well studied ; moreover , due to the heavy burden of psychological disturbance , psychiatric morbidity , and disability in daily life , the mental health of copd patients has received growing attention in recent years.3,4 the prevalence of cognitive impairment in patients with copd ranges from 12% to 88%5 and is associated with depression , poor quality of life , which may affect patients ability to manage their disease , and reduced compliance with medication and oxygen therapy,6,7 leading to adverse clinical outcomes.811 therefore , cognitive function as well as emotional function are important aspects of the overall clinical care of patients with copd . it has been suggested that a multidimensional assessment and personalized disease management approach could be an optimal strategy for addressing comorbidities , self - management education , and risk factor modification in copd patients.7 the early identification of cognitive dysfunction is critical if outcomes are to be improved in this population , and an understanding of the characteristics associated with a higher risk of cognitive impairment may assist health care professionals to address this challenge . the primary aim of this study was to examine the cross - sectional prevalence of cognitive impairment in an unselected population of copd patients ; the secondary objective was to determine the relationships between cognitive impairment and major demographic and clinical variables , such as lung function , depression , and quality of life . the present study is a post hoc analysis of a cross - sectional , observational study conducted in respiratory medicine departments and primary care centers in spain.11 this study was aimed at examining the prevalence of depressive symptoms and moderate - to - severe depression in copd and included ambulatory patients who were 40 years of age or older , with stable copd ( confirmed by postbronchodilator forced expiratory volume in 1 second / forced vital capacity < 70% and absence of exacerbations in the previous 3 months ) . all patients that completed the mini - mental state examination ( mmse ) were included in this post hoc analysis . the study was approved by the institutional ethics committee of the hospital clinic ( barcelona , spain ) and was conducted in accordance with the principles of the declaration of helsinki . prior to participation , all patients signed written documentation that the informed consent process was completed . investigators recorded patients sociodemographic data and clinical information on copd severity using the modified medical research council dyspnea scale12 and the body mass index , airflow obstruction , dyspnea and exacerbations ( bodex ) index,13 comorbidity according to the charlson index,14 exacerbations in the previous year , and treatment . this instrument explores spatial and temporal orientation , registration , attention , and calculation , recall , language , and visual construction in 12 items and 30 questions . a correct answer to one question a score of less than 27 indicated cognitive impairment.17 depressive symptoms were measured using the short beck depression inventory questionnaire.18,19 this tool is a 13-item self - administered inventory that assesses affective , cognitive , motivational , and vegetative symptoms of depression . items use a 4-point scale that ranges from 0 to 3 and a total score is calculated by adding up the item responses . health - related quality of life was assessed by the generic euroqol-5 dimensions ( eq-5d ) questionnaire and the specific copd assessment test ( cat ) . the eq-5d consists of a 5-item descriptive system ( including mobility , self - care , usual activities , pain / discomfort , and anxiety / depression ) , with each item rated as no problems , some problems , or severe problems , and an overall health state score for the day of the assessment , measured on a visual analog scale ( 0100 ; 100 represents best overall health).20 the cat is a short , specific quality of life questionnaire for measuring the impact of copd on the patient s well - being and daily life . it consists of eight items , each presented as a 6-point semantic differential scale , providing a score out of 40 , indicating the impact of the disease.21 we used the validated spanish version of cat.22 physical activity was measured by asking patients how many minutes , on average , they walked every weekday , as previously described.23,24 depending on their daily walking activity , patients were classified into three groups : patients who walked < 30 minutes , patients who walked between 30 and 60 minutes , and patients who walked 60 minutes . fisher s exact test was used for comparisons of qualitative variables , with the bonferroni correction for all pairwise comparisons . student s t - test was used to determine the relationship between qualitative variables by group . odds ratio ( or ) univariates were calculated by logistic regression to evaluate the different risks contemplated in the study , including all demographic , clinical , and questionnaires variables . the first model was developed with cognitive status as a dependent variable and all variables that showed a significant association with cognition in univariate analysis as independent variables ( model 1 ) . a second model ( model 2 ) excluded any variables derived from the use of the questionnaires from the independent variables . the objective of model 2 was to identify the factors associated with cognitive impairment that could be identified in routine clinical practice without the administration of questionnaires . statistical analyses were performed using sas version 9.1.3 service pack 3 software ( sas institute inc . , cary , nc , usa ) . the present study is a post hoc analysis of a cross - sectional , observational study conducted in respiratory medicine departments and primary care centers in spain.11 this study was aimed at examining the prevalence of depressive symptoms and moderate - to - severe depression in copd and included ambulatory patients who were 40 years of age or older , with stable copd ( confirmed by postbronchodilator forced expiratory volume in 1 second / forced vital capacity < 70% and absence of exacerbations in the previous 3 months ) . all patients that completed the mini - mental state examination ( mmse ) were included in this post hoc analysis . the study was approved by the institutional ethics committee of the hospital clinic ( barcelona , spain ) and was conducted in accordance with the principles of the declaration of helsinki . prior to participation , all patients signed written documentation that the informed consent process was completed . investigators recorded patients sociodemographic data and clinical information on copd severity using the modified medical research council dyspnea scale12 and the body mass index , airflow obstruction , dyspnea and exacerbations ( bodex ) index,13 comorbidity according to the charlson index,14 exacerbations in the previous year , and treatment . this instrument explores spatial and temporal orientation , registration , attention , and calculation , recall , language , and visual construction in 12 items and 30 questions . a correct answer to one question a score of less than 27 indicated cognitive impairment.17 depressive symptoms were measured using the short beck depression inventory questionnaire.18,19 this tool is a 13-item self - administered inventory that assesses affective , cognitive , motivational , and vegetative symptoms of depression . items use a 4-point scale that ranges from 0 to 3 and a total score is calculated by adding up the item responses . health - related quality of life was assessed by the generic euroqol-5 dimensions ( eq-5d ) questionnaire and the specific copd assessment test ( cat ) . the eq-5d consists of a 5-item descriptive system ( including mobility , self - care , usual activities , pain / discomfort , and anxiety / depression ) , with each item rated as no problems , some problems , or severe problems , and an overall health state score for the day of the assessment , measured on a visual analog scale ( 0100 ; 100 represents best overall health).20 the cat is a short , specific quality of life questionnaire for measuring the impact of copd on the patient s well - being and daily life . it consists of eight items , each presented as a 6-point semantic differential scale , providing a score out of 40 , indicating the impact of the disease.21 we used the validated spanish version of cat.22 physical activity was measured by asking patients how many minutes , on average , they walked every weekday , as previously described.23,24 depending on their daily walking activity , patients were classified into three groups : patients who walked < 30 minutes , patients who walked between 30 and 60 minutes , and patients who walked 60 minutes . fisher s exact test was used for comparisons of qualitative variables , with the bonferroni correction for all pairwise comparisons . student s t - test was used to determine the relationship between qualitative variables by group . odds ratio ( or ) univariates were calculated by logistic regression to evaluate the different risks contemplated in the study , including all demographic , clinical , and questionnaires variables . the first model was developed with cognitive status as a dependent variable and all variables that showed a significant association with cognition in univariate analysis as independent variables ( model 1 ) . a second model ( model 2 ) excluded any variables derived from the use of the questionnaires from the independent variables . the objective of model 2 was to identify the factors associated with cognitive impairment that could be identified in routine clinical practice without the administration of questionnaires statistical analyses were performed using sas version 9.1.3 service pack 3 software ( sas institute inc . , cary , nc , usa ) . of 1,273 screened patients , 333 had not had the mmse evaluation and were excluded from the analysis . both populations ( excluded and included ones ) included patients were predominantly male , with an age ranging from 40 to 90 years . a total of 370 patients ( 39.4% ) scored below the threshold of 27 for suspicion of mild cognitive impairment ( mci ; figure 1 ) . patients with mmse scores < 27 were older , had a lower level of formal education , had heavier smoking exposure , presented more respiratory symptoms , had more frequently domiciliary oxygen , suffered a higher number of and more severe exacerbations during the previous year , and had a greater comorbidity burden . in addition , higher levels of depression , more suicidal ideation , poorer quality of life , and less physical activity were more frequent in cognitively impaired patients . in the univariate analysis , cognitive impairment was associated with higher age , lower educational level , worse lung function and more severe copd , more frequent respiratory symptoms and exacerbations , and oxygen therapy requirement . higher comorbidity burden , more depressive symptoms , and more impaired health status ( eq-5d and cat ) were also associated with cognitive impairment . higher levels of physical activity were associated with better cognitive function ( table 2 ) . in the multivariate analysis , only educational level and the eq-5d social tariff were independently and significantly associated with cognitive impairment ( table 2 ) . when the analysis was limited to the usual demographic and clinical variables , and the questionnaires were excluded from the model , the significant factors in the multivariate analysis were educational level , history of exacerbations , the bodex index , and the charlson comorbidity index ( table 3 ) . of 1,273 screened patients , 333 had not had the mmse evaluation and were excluded from the analysis . both populations ( excluded and included ones ) included patients were predominantly male , with an age ranging from 40 to 90 years . a total of 370 patients ( 39.4% ) scored below the threshold of 27 for suspicion of mild cognitive impairment ( mci ; figure 1 ) . patients with mmse scores < 27 were older , had a lower level of formal education , had heavier smoking exposure , presented more respiratory symptoms , had more frequently domiciliary oxygen , suffered a higher number of and more severe exacerbations during the previous year , and had a greater comorbidity burden . in addition , higher levels of depression , more suicidal ideation , poorer quality of life , and less physical activity were more frequent in cognitively impaired patients . in the univariate analysis , cognitive impairment was associated with higher age , lower educational level , worse lung function and more severe copd , more frequent respiratory symptoms and exacerbations , and oxygen therapy requirement . higher comorbidity burden , more depressive symptoms , and more impaired health status ( eq-5d and cat ) were also associated with cognitive impairment . higher levels of physical activity were associated with better cognitive function ( table 2 ) . in the multivariate analysis , only educational level and the eq-5d social tariff were independently and significantly associated with cognitive impairment ( table 2 ) . when the analysis was limited to the usual demographic and clinical variables , and the questionnaires were excluded from the model , the significant factors in the multivariate analysis were educational level , history of exacerbations , the bodex index , and the charlson comorbidity index ( table 3 ) . copd is a multicomponent inflammatory disease that affects physical and nonphysical functions , one of which is cognition . in our study , we found some degree of cognitive impairment in almost 40% of unselected copd patients , as determined by a mmse score < 27 . previous estimates of cognitive dysfunction in copd patients ranged from 12% to 88% , depending on the study population and the tools used for neuropsychological assessment.5,9 a recent study in a very similar population ( including not only elderly patients , but also young patients ) to ours17 found mci in 36% of copd patients after comprehensive neuropsychological testing . the authors reported that a mmse score of 27 provided optimal maximum accuracy and a diagnostic cutoff ( < 27 indicated impairment ) , with 97% specificity and 73% of patients correctly classified . however , they also found that the montreal cognitive assessment performed better as a screening test for detecting mci in patients with copd . so , this threshold for mild impairment is not necessarily indicative of a clinically significant cognitive decline associated with functional impairment , but could serve as an early identification of an affected cognitive function . cognitive symptoms are core symptoms in many mental disorders;25 they impact on cognitive functioning , which deteriorates as patients get older , particularly after the age of 60.26 copd patients score lower on standard cognitive performance tests over time , compared with individuals of the same age without the disease.27 one hypothesis for this accelerated decline in copd patients is altered brain perfusion.28 changes in brain perfusion due to hypoxemia in subjects with severe copd may increase cognitive impairment.29 in our population , more cognitively affected patients were receiving domiciliary oxygen that could be a correlate of higher disease severity and sustained periods of hypoxemia . in contrast , a recent study indicated that long - term home oxygen therapy allowed to preserve cognitive functions from the copd - induced deterioration,30 so the role of supplemental oxygen in preventing copd - induced cognitive deterioration is still controversial . indeed , other factors , such as hypercapnia or oxidative stress , may also be involved.31 other authors have suggested that intermittent and continuous hypoxia resulting from poor lung function may lead to transient deficits in neurotransmitter metabolism in the central nervous system.3234 several mechanisms , then , appear to be involved in cognitive decline in this population . prospective neuroimaging studies are required to characterize brain changes and corresponding disturbances in cognitive function in these patients over time . cognitive impairment represents a critical health care burden in terms of costs.35 individuals with mci have a higher risk of developing dementia than the general population.36 moreover , cognitive dysfunction in copd has been associated with poorer outcomes and even with an increase in disability and mortality.8,37,38 thus , it is essential that this condition is identified early in copd patients , in order to prevent or delay progression to clinical dementia or increased morbidity . if the factors affecting cognition are recognized , cognitive impairment may be detected earlier and copd patients at higher risk may be identified . in our study , the multivariate analysis showed that educational level and quality of life assessed by eq-5d were significantly and independently associated with the presence of cognitive impairment . however , the use of health - related quality of life or other questionnaires is not a routine practice in most primary care offices , so we ran a multivariate model , discarding the scores of these questionnaires and including only demographic and clinical variables . in this model , cognitive impairment was associated with exacerbations in the previous year , the severity of copd measured by the bodex index , and a higher comorbidity burden . they have been associated with increased health care costs,39 a significant decline in health status,40 and substantial mortality.41 our results suggest that frequent exacerbations also have negative consequences on cognitive function . this is in line with a previous study that investigated the cognitive function of copd patients who were hospitalized following an acute exacerbation . these patients had significantly poorer cognitive function compared with control participants 3 months after discharge from the hospital.10 other studies have shown that cognitive impairment during the exacerbation period resolves during periods of stability.4244 more exacerbations and increased copd severity could reflect the poorer compliance with medication associated with cognitive impairment.6,7 the systemic inflammation seen in severe copd and during acute exacerbations45 may participate in neurocognitive impairment via a direct neurotoxic effect or by affecting cerebral atherosclerosis.9 a high comorbidity burden may also contribute to persistent brain injury : multiple concomitant diseases , such as cerebrovascular disease and related mechanisms , including endothelial dysfunction and oxidation , may lead to neuronal death , synaptic dysfunction , and cognitive impairment.46 all these data confirm that the cognitive impairment that occurs in copd patients is associated with disease severity . neuropsychiatric assessment should become a routine part of the diagnostic procedure for these patients , to help physicians grade the overall impact of copd and determine the most effective treatment and strategies . the cross - sectional design of our study limits any type of causal inferences , and the directionality between copd and cognition remains unclear . despite the frequent use of the mmse in clinical research and practice , this tool for cognitive impairment detection could have missed key domains of cognition often affected in copd ( ie , executive functioning ) . an additional limitation of our study is the lack of data on our patients medical treatment . as treatment may have great impact on the symptom burden and mental health of the patients , such information should be collected in future studies . in particular , prospective studies are urgently needed to determine the most effective behavioral and medical interventions for reducing the risk of poor neurocognitive outcomes in patients with copd . early detection of cognitive decline is crucial , in view of its association with poorer copd outcomes , including increased mortality , and patients with frequent exacerbations and or when the bodex index appears concerning the clinician may want to ask the patient and/or caregivers about cognitive function . these patients may need more individualized educational and care interventions to help them manage their daily lives . clinicians must involve family caregivers in the care plan of patients with severe copd and cognitive deficits .

to date , a range of ion channels have been identified in chondrocytes using a number of different techniques , predominantly electrophysiological and/or biomolecular ; each of these has its advantages and disadvantages . here we aim to compare and contrast the data available from biophysical and microarray experiments . this letter analyses recent transcriptomics datasets from chondrocytes , accessible from the european bioinformatics institute ( ebi ) . we discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes . the ion channels which appear most frequently across these microarray datasets are discussed , along with their possible functions . we discuss whether functional or protein data exist which support the microarray data . a microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes , namely the genes encoding large calcium - activated potassium ( bk ) and aquaporin channels . purposewe investigated the association between cognitive impairment and chronic obstructive pulmonary disease ( copd ) , taking into account demographic and clinical variables evaluated during routine practice.patients and methodswe performed a post hoc analysis of a cross - sectional study that included subjects with stable copd . sociodemographic and clinical information was recorded using the body mass index , airflow obstruction , dyspnea and exacerbations index and the charlson comorbidity index . cognitive performance was studied by the mini - mental state examination , with a score less than 27 indicating clinical impairment . depressive symptoms , physical activity , and quality of life ( euroqol-5 dimensions and copd assessment test ) were also evaluated.resultsthe analysis included 940 subjects . the prevalence of cognitive impairment was 39.4% . multivariate logistic regression models revealed that cognitive impairment was associated with educational level ( odds ratio [ or ] = 0.096 , 95% confidence interval [ ci ] = 0.0110.447 ) and poorer quality of life measured by the euroqol-5 dimensions social tariff ( or = 0.967 , 95% ci = 0.9500.983 ) . when questionnaires were not included in the analysis , cognitive impairment was associated with educational level ( or = 0.063 , 95% ci = 0.0100.934 ) , number of exacerbations ( or = 11.070 , 95% ci = 1.45084.534 ) , body mass index , airflow obstruction , dyspnea and exacerbations index score ( or = 1.261 , 95% ci = 1.0491.515 ) , and the charlson comorbidity index ( or = 1.412 , 95% ci = 1.1181.783).conclusioncognitive impairment is common in copd and is associated with low educational level , higher disease severity , and increased comorbidity . this could have therapeutic implications for this population .

intestinal helminths are among the most common and widespread of human infections , contributing to poor nutritional status , anemia and impaired growth ( 1 ) . intestinal helminthiases are also known to aggravate pre - existing anemia by decreasing appetite and thus food and iron intake ( 2 , 3 ) . worldwide , anemia is an important reproductive health problem because of its association with adverse pregnancy outcome such as increased rates of maternal and perinatal mortality , premature delivery , low birth weight , etc ( 4 ) . women in developing countries spend half of their reproductive lives pregnant and lactating and a high proportion of women in developing countries become anemic during this period . women of reproductive age who are iron deficient but not anemic may become anemic during pregnancy as a consequence of increased iron requirements and expanded plasma volume . epidemiological surveys have revealed that poor sanitation and inappropriate environmental conditions coupled with indiscriminate defaecation , geophagy and contamination of water bodies are the most important predisposing factors to intestinal worm infection ( 5 ) . practices such as hand washing , disposal of refuse , personal hygiene , wearing of shoes and others , when not done properly may contribute to the infection or picking of these worms from the environments ( 6 ) . this research investigates the prevalence of helminth infection and its hematological alterations during pregnancy findings of this study will serve as a tool in evidence based health education on the need to intensify efforts at preventing helminthiases and its attendant risk of anemia during pregnancy . two hundred and eighty - two pregnant women between the ages of 18 45 years , in their various trimesters and of various parities ( 0 10 ) were enlisted . fresh stool samples for helminth screening were collected from each of the 282 subjects in dry , clean , leak proof and sterilized sample containers . the samples were examined for consistency and presence of cysts , proglottids and adult worms . concentrated saturated sodium chloride floatation and formol - ether concentration techniques were used for fecal analysis . the total number of eggs was counted under x40 magnification of a compound microscope stool samples were processed within 8 hours of collection and examined microscopically within one hour of preparation to avoid over clearance of hookworm ova . based on the thresholds recommended by the world health organization ( who ) , helminth intensities were classified as light , moderate or severe ( 7 ) . using a sterile syringe , 3mls of venous blood was collected from each of the subjects and transferred into a capillary tube . the pcv of each specimen was determined using a hewkley microhematocrit reader and classified as follows : mild ( pcv 2729% ) , moderate ( pcv 1926% ) , and severe ( pcv below 19% ) . 4g / dl- very severe anemia , hb < 8g / dl- severe anemia , hb < 9g / dl- moderate anemia and hb < 11g / dl- mild anemia. data entry and validation was performed in excel , and statistical analysis was done using statistical package for social sciences ( spss ) version 17.0 . values were considered statistically significant when p - values were less than 0.05 ( p<0.05 ) . pearson chi - square , t - test and correlations were used to determine the association between hemoglobin concentrations and helminth infection as indicators of anemia . at the onset of the study , thereafter , they were given informed consent forms to sign for their communities and households after their contents were translated to them in local languages . two hundred and eighty - two pregnant women between the ages of 18 45 years , in their various trimesters and of various parities ( 0 10 ) were enlisted . fresh stool samples for helminth screening were collected from each of the 282 subjects in dry , clean , leak proof and sterilized sample containers . the samples were examined for consistency and presence of cysts , proglottids and adult worms . concentrated saturated sodium chloride floatation and formol - ether concentration techniques were used for fecal analysis . the total number of eggs was counted under x40 magnification of a compound microscope stool samples were processed within 8 hours of collection and examined microscopically within one hour of preparation to avoid over clearance of hookworm ova . based on the thresholds recommended by the world health organization ( who ) , helminth intensities were classified as light , moderate or severe ( 7 ) . using a sterile syringe , 3mls of venous blood was collected from each of the subjects and transferred into a capillary tube . the pcv of each specimen was determined using a hewkley microhematocrit reader and classified as follows : mild ( pcv 2729% ) , moderate ( pcv 1926% ) , and severe ( pcv below 19% ) . 4g / dl- very severe anemia , hb < 8g / dl- severe anemia , hb < data entry and validation was performed in excel , and statistical analysis was done using statistical package for social sciences ( spss ) version 17.0 . values were considered statistically significant when p - values were less than 0.05 ( p<0.05 ) . pearson chi - square , t - test and correlations were used to determine the association between hemoglobin concentrations and helminth infection as indicators of anemia . at the onset of the study , the community and household heads were well briefed on the objectives of the study . thereafter , they were given informed consent forms to sign for their communities and households after their contents were translated to them in local languages . the gastrointestinal helminth parasites observed in this study were hookworm ( 8.5% ) , ascaria lumbricoides ( 5.0% ) and trichuris trichiura ( 0.7% ) , while mixed infection accounted for 2.1% ( table 1 ) . of the 282 pregnant women examined , 46(16.3% ) were infected with at least one parasite species . age specific prevalence showed that subjects of 18 20 years age group had the highest rate of infection ( 27.0% ) while those of 4145 years had the least rate ( 0% ) . the difference in infection by age groups was statistically significant ( p<0.05 , = 28.759 , df=12 ) . within the trimester , pregnant women in their first trimester had the highest infection rate of 20.9% while those in their third trimester had the least ( 12.9% ) . the differences were however not statistically significant ( p>0.05 ; = 6.895 , df = 8) . the primigravidae had the highest prevalence ( 27.5% ) while the gravidae 7 group had the least rate ( 7.7% ) . differences in the prevalence of helminth infections by parity groups was statistically significant ( p<0.05 ; = 32.437 , df=12 ) . the intensity of infection among pregnant women ( table 2 ) shows that 24(80.0% ) of the pregnant women examined had single ( hookworm ) infection while 6 ( 20.0% ) had mixed ( hookworm and a. lumbricoides ) infection . fifteen ( 62.5% ) of the women with hookworm infection had light infection while 5(20.8% ) and 4(16.7% ) had moderate and heavy infections respectively . out of the pregnant women with mixed infection , 4 ( 66.7% ) had light infection while 2(33.3% ) had moderate infection . also , 14 ( 70.0% ) of infected women had a. lumbricoides infection , of which 8(57.1% ) had light , 4(28.6% ) moderate and 2(14.3% ) heavy infections , respectively . only two women were positive to t. trichiura infection , one of which is light while the other moderate . the differences in the intensities of infection was not statistically significant ( = 0.967 , df=2 , p>0.05 ) a total of 166(58.9% , meansd = 9.31.0 ) of the pregnant women were anaemic . out of these , 92(32.6% ) had mild anemia , 58(20.6% ) had moderate anemia while 16(5.7% ) had severe anemia ( table 3 ) . age specific prevalence shows that women between the ages of 31 40 years were most anemic with prevalence rate of 71.7% , meansd = 9.11.1 ) while those of 1820 years had the least ( 18(48.6% , meansd = 9.21.0 ) - table 3 . the differences in the hemoglobin levels of these age groups was statistically significant ( p<0.05 , = 17.197 , df=9 ) . table 3 also shows that the pregnant women in their second trimester ( 63.6% and meansd = 9.31.0 ) had the most severe anemia . the difference in the hemoglobin levels by trimester groups was not statistically significant ( p > 0.05 , = 2.794 , df=6 , meansd=9.31.0 ) . women in their secondigravidae had the most severe anemia ( 61.8% , meansd = 9.30.9 ) . the differences in hemoglobin levels by parity was not statistically significant ( p>0.05 , = 9.034 , df=6 , meansd = 9.31.0 ) table 3 . pregnant women who were infected with one helminth or the other were observed to have lower mean hemoglobin ( hb ) of 8.600.22g / dl than that of the uninfected ( 9.720.07g / dl ) . significant difference ( t - value = 5.660 , p<0.05 ) was observed between the hb of the infected and uninfected pregnant women . in addition , pregnant women infected with one helminth or the other had a mean pcv of 26.090.65% while the uninfected had 34.542.96% . the mean pcv of infected pregnant women was also significantly different ( t - value= 0.013 , p<0.05 ) from that of uninfected pregnant women . the correlation between hb , pcv and helminth infections hookworm infection was observed to have a moderate highly significant negative correlation with hb ( r= 0.389 , p<0.01 ) and pcv ( r= 0.277 , p<0.01 ) . mixed infections ( hookworm and ascaris lumbricoides ) were also observed to have a mild highly significant negative correlation with hb ( r=0.179 , p<0.01 ) and pcv ( r=0.192 , p<0.01 ) . furthermore , a. lumbricoides and t. trichiura infections were observed to have a negative correlation with hb and pcv respectively but were not statistically significant ( p>0.05 ) . the gastrointestinal helminth parasites observed in this study were hookworm ( 8.5% ) , ascaria lumbricoides ( 5.0% ) and trichuris trichiura ( 0.7% ) , while mixed infection accounted for 2.1% ( table 1 ) . of the 282 pregnant women examined , 46(16.3% ) were infected with at least one parasite species . age specific prevalence showed that subjects of 18 20 years age group had the highest rate of infection ( 27.0% ) while those of 4145 years had the least rate ( 0% ) . the difference in infection by age groups was statistically significant ( p<0.05 , = 28.759 , df=12 ) . within the trimester , pregnant women in their first trimester had the highest infection rate of 20.9% while those in their third trimester had the least ( 12.9% ) . the differences were however not statistically significant ( p>0.05 ; = 6.895 , df = 8) . the primigravidae had the highest prevalence ( 27.5% ) while the gravidae 7 group had the least rate ( 7.7% ) . differences in the prevalence of helminth infections by parity groups was statistically significant ( p<0.05 ; = 32.437 , df=12 ) . the intensity of infection among pregnant women ( table 2 ) shows that 24(80.0% ) of the pregnant women examined had single ( hookworm ) infection while 6 ( 20.0% ) had mixed ( hookworm and a. lumbricoides ) infection . fifteen ( 62.5% ) of the women with hookworm infection had light infection while 5(20.8% ) and 4(16.7% ) had moderate and heavy infections respectively . out of the pregnant women with mixed infection , 4 ( 66.7% ) had light infection while 2(33.3% ) had moderate infection . also , 14 ( 70.0% ) of infected women had a. lumbricoides infection , of which 8(57.1% ) had light , 4(28.6% ) moderate and 2(14.3% ) heavy infections , respectively . only two women were positive to t. trichiura infection , one of which is light while the other moderate . the differences in the intensities of infection was not statistically significant ( = 0.967 , df=2 , p>0.05 ) . out of these , 92(32.6% ) had mild anemia , 58(20.6% ) had moderate anemia while 16(5.7% ) had severe anemia ( table 3 ) . age specific prevalence shows that women between the ages of 31 40 years were most anemic with prevalence rate of 71.7% , meansd = 9.11.1 ) while those of 1820 years had the least ( 18(48.6% , meansd = 9.21.0 ) - table 3 . the differences in the hemoglobin levels of these age groups was statistically significant ( p<0.05 , = 17.197 , df=9 ) . table 3 also shows that the pregnant women in their second trimester ( 63.6% and meansd = 9.31.0 ) had the most severe anemia . the difference in the hemoglobin levels by trimester groups was not statistically significant ( p > 0.05 , = 2.794 , df=6 , meansd=9.31.0 ) . women in their secondigravidae had the most severe anemia ( 61.8% , meansd = 9.30.9 ) . the differences in hemoglobin levels by parity was not statistically significant ( p>0.05 , = 9.034 , df=6 , meansd = 9.31.0 ) table 3 . pregnant women who were infected with one helminth or the other were observed to have lower mean hemoglobin ( hb ) of 8.600.22g / dl than that of the uninfected ( 9.720.07g / dl ) . significant difference ( t - value = 5.660 , p<0.05 ) was observed between the hb of the infected and uninfected pregnant women . in addition , pregnant women infected with one helminth or the other had a mean pcv of 26.090.65% while the uninfected had 34.542.96% . the mean pcv of infected pregnant women was also significantly different ( t - value= 0.013 , p<0.05 ) from that of uninfected pregnant women . hookworm infection was observed to have a moderate highly significant negative correlation with hb ( r= 0.389 , p<0.01 ) and pcv ( r= 0.277 , p<0.01 ) . mixed infections ( hookworm and ascaris lumbricoides ) were also observed to have a mild highly significant negative correlation with hb ( r=0.179 , p<0.01 ) and pcv ( r=0.192 , p<0.01 ) . furthermore , a. lumbricoides and t. trichiura infections were observed to have a negative correlation with hb and pcv respectively but were not statistically significant ( p>0.05 ) . the prevalence of intestinal helminth infections among the study population ( 16.3% ) is epidemiologically significant considering the fact that this is an epidemiological survey involving asymptomatic subjects . it has been observed that any helminth ova or larvae present would be in very low level and possibly undetectable ( 9 ) . the high prevalence of hookworm infection compared to the a. lumbricoides and t. trichiura infections may be attributed to the cultural practices of the subjects especially agriculture and also high level of unhygienic practices . the prevalence of parasitic infections among pregnant women differed significantly ( p<0.05 ) within the age groups , indicating gestational - age dependence . findings in this study show that pregnant women in their first trimester were more infected than those in second and third trimesters . this can be attributed to the fact that treatment of helminthiases during ante natal visits is done after the first trimester . that is , pregnant women are given anthelminthic drugs after their first trimester ( 11 ) . when gestational age was related to anemia , women in their second pregnancy trimester were more anemic than their counterparts in their first and third trimesters . however , anemia in many areas of africa was described as usually most severe in the second trimester of gestation , especially following a period of acute infection , e.g. malaria , in the first trimester ( 12 , 13 ) . this study established an association between the intensity of helminth infections and lower hemoglobin ( hb ) . pregnant women with light infections were found to have low hemoglobin levels , but women with heavy infections had lower hemoglobin levels . the pathenogenicity of helminth infection shows that the disease manifests in three main phases , with the intestinal phase representing the most important period . a moderate hookworm infection according to studies will gradually produce anemia as the body reserves of iron are used up , with the severity depending on the worm load and the dietary intake of iron ( 12 ) . the burden of disease imposed on helminth - infected girls and women of childbearing age , especially when pregnant , may very well define the single most important contribution of intestinal parasitic infections to the calculation of their global disease burden . this study reveals a significant difference ( p<0.05 ) in the mean hb and pcv of the infected and uninfected pregnant women . pregnant women who were infected with at least one helminth parasite presented not just a higher frequency of anemia but also significant lower level of hemoglobin and pcv . ascaris lumbricoides and t. trichiura infections were also observed to have a negative correlation with hb and pcv among infected pregnant women but were not significant ( p>0.05 ) . public health importance where its prevalence is between 20% and 39.9% and severe if it occurs in 40% or more of the population . given these results , the importance and potential impacts of intestinal helminthiases during pregnancy , such as anemia , this indicates the need for periodical stool examinations during pregnancy as part of routine laboratory test in the prenatal control of helminthiases . a single course of anthelminthic therapy in addition to iron - folate supplementation would significantly increase hemoglobin concentrations and improve iron status in pregnant women . as has been stated in other studies , it is necessary to modify some preventive measures of information and education and to give specific treatment before the pregnancy in order to increase some of the pregnant women s health indicators . also , anthelmintic therapy which is inexpensive and safe during pregnancy after the first trimester should be part of the antenatal programme since malaria diagnosis and treatment is also part of the antenatal programme ( 14 ) . this study established an association between the intensity of helminth infections and lower hemoglobin ( hb ) . there is need for periodical stool examinations during pregnancy as part of routine laboratory test in the prenatal control of intestinal helminth infection . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors . soft tissue sarcomas are very rare tumors ; however , there are many histologic subtypes . among those subtypes , furthermore , among sarcomas originating within the retroperitoneum , which constitute 1015% of all soft tissue sarcomas , liposarcomas are the most common histologic type , accounting for 41% of these tumors [ 2 , 3 ] . liposarcomas are generally located in the head , neck , trunk , mediastinum , upper and lower extremities , gastrointestinal tract , and retroperitoneum . they commonly occur in patients aged 4060 years , and men and women are equally affected . the dimensions and weight of liposarcomas are variable ; those over 20 kg are called herein , we report a giant retroperitoneal liposarcoma weighing 25.0 kg , encasing the entire left kidney and adherent to adjacent structures which was successfully removed with kidney and aorta preserving . the patient had not experienced any symptoms , such as abdominal pain , nausea , vomiting , constipation , dyspepsia , or dyspnea . the patient was admitted to the hospital and underwent contrast - enhanced computed tomography ( ct ) of the abdomen . the scan revealed a huge fatty mass originating from the retroperitoneum probably indicative of retroperitoneal liposarcoma . the spleen was pushed anteriorly and the small bowel was deviated to the right side of the intra - abdominal space by the mass ( fig . 1 ) . because the patient was old , we decided to attempt organ - preserving surgery for the removal of the tumor to minimize the morbidity . adherence of the mass to the diaphragm , stomach , spleen , pancreas , and aorta could be observed . the greatest difficulty was that the tumor was encasing the entire left kidney and adherent to the aorta . although the entire left kidney was encased with the huge tumor , neither the renal parenchyma nor the ureter was invaded . we successfully performed a salvage of the left kidney by wide excision and separated the tumor from the aorta by shaving it away , thus preserving both kidney and the aorta . the specimen measured 45.0 30.0 11.0 cm and weighted 25.0 kg ( fig . 2 ) . microscopic examination showed a combined type of liposarcoma ( tumor component : well - differentiated liposarcoma , more than 95% , myxoid liposarcoma , less than 5% ) . an average of 12 mitotic figures were noted per high - power field . according to the grading system of the french federation of cancer centers sarcoma group , she underwent regular follow - up examinations for 16 months after the operation . at 16 months , a follow - up ct scan revealed a newly defined low - density soft tissue mass in the aortocaval and preaortocaval area , measuring 3.5 1.8 4.3 cm , suggesting a locally recurrent tumor in the retroperitoneum . we performed positron emission tomography ( pet ) , which revealed tumor recurrence in the retroperitoneum . fluorescence in situ hybridization showed mdm2 amplification , which was consistent with a diagnosis of well - differentiated liposarcoma . after the second surgery , the patient underwent regular follow - up ct scans for approximately 12 months , and to date , there has been no evidence of tumor recurrence liposarcoma is one of the most common soft tissue sarcomas , constituting approximately 20% of cancers within that group . between 10 and 15% of soft tissue sarcomas originate within the retroperitoneal space , and the most common type among these is liposarcoma . however , liposarcoma is very rare overall , accounting for 0.20.3% of all malignancies . multiple factors , such as site and depth of origin , margin involvement after resection , and histologic grade affect survival rates for patients with liposarcoma . if the size of the tumor is less than 2.5 cm , the rate of metastasis at 5 years is approximately 3% . on the other hand , the rate of metastasis at 5 years is between 55 and 60% in cases of tumors larger than 20 cm . this is because the retroperitoneal space allows the tumor to grow to a large size before the appearance of clinical signs and symptoms . therefore , the tumor is often diagnosed at the advanced stage . resection margin involvement also affects prognosis . the five recognized histologic types are the well - differentiated , myxoid , round cell , pleomorphic , and dedifferentiated type . the well - differentiated type has good prognosis , with 5-year survival rates of approximately 90% . however , the toxic effects of radiation therapy limit this option by primary treatment modality . research has documented little benefit from adjuvant chemotherapy in well - differentiated low - grade tumors , and partial responses in high - grade diseases in up to 50% of patients , with increased overall survival . as a result , complete surgical resection is the gold standard treatment , which might be curative . in many cases , combined resection of involved organs and vasculatures therefore , the most commonly sacrificed organ is the kidney , followed by the colon , pancreas , major vasculature , and spleen . in this study , we reported on a giant retroperitoneal liposarcoma encasing the entire left kidney and adherent to adjacent structures . furthermore , we described successful organ - preserving surgical removal and discussed the prognosis . although there has been no evidence of recurrence to date , we will continue to observe our patient closely for recurrence , as in other previously published reports .

backgroundthe incidence and hematological effects of helminth infection during pregnancy were investigated among pregnant women in isiala , mbano , southeast nigeria.methods:totally 282 pregnant women were enlisted for the study between october 2011 and september 2012 . stool samples were examined for intestinal helminths using formalin - ether sedimentation technique . hemoglobin ( hb ) and packed cell volume ( pcv ) levels were evaluated in venous blood samples using sahli s and microhaematocrit methods respectively.results:forty six ( 16.3% ) subjects were infected with at least one helminth parasite ; 24 ( 8.5% ) hookworm , 14(5.0% ) and 2(0.7% ) a. lumbricoides and trichuris trichiura infections respectively . intestinal helminthiases in pregnant women was significantly associated with age ( p<0.05 ) . the prevalence of intestinal helminthiases by parity was also significantly different ( p<0.05 ) with primigravidae having the highest infection rate ( 27.5% ) . hematological assessment showed that the prevalence of anemia among the women was 58.9% ( meansd = 9.31.0 ) . the differences in hemoglobin levels by age groups was statistically significant ( p < 0.05 ) . the contributory effect of gastrointestinal helminths in anemia showed that infected pregnant women had lower mean hemoglobin ( 8.600.22g / dl ) than the uninfected ( 9.720.07g / dl ) . significant difference ( t - value = 5.660 , p<0.05 ) was observed between the hb of the infected and uninfected pregnant women . in addition , infected pregnant women had mean pcv of 26.090.65% while the uninfected had 34.542.96% . the mean pcv of infected pregnant women was significantly different ( t - value= 0.013 , p<0.05 ) from that of the uninfected.conclusionanti-helminthic therapy after the first trimester should be part of the antenatal programme . intestinal helminth infection showed significant negative correlation with hb and pcv and contributed moderately to anemia . retroperitoneal liposarcoma is a rare tumor . the dimension and weight of liposarcoma are variable ; those over 20 kg are called giant liposarcoma. herein , we report giant retroperitoneal liposarcoma measuring 45 cm in diameter and 25 kg in weight encasing the entire left kidney and adherent to adjacent structures . a 71-year - old woman presented for a regular checkup . image study revealed a huge mass probably indicative of retroperitoneal liposarcoma encasing the entire left kidney and adherent to adjacent structures . we performed an organ - preserving surgical removal . the pathologic report was liposarcoma . at postoperative month 16 , a follow - up ct revealed a locally recurrent tumor . the patient underwent surgical removal of the newly discovered mass . after the second surgery , the patient underwent regular follow - up ct for approximately 12 months , and to date , there has been no evidence of tumor recurrence . high - grade liposarcoma shows sensitivity to radiation therapy . however , the toxic effect of radiation therapy limits this option by treatment modality . the use of chemotherapy is also controversial . as a result , complete resection is the gold standard treatment . here , we report a giant retroperitoneal liposarcoma encasing the entire left kidney and adherent to adjacent structures , describe successful organ - preserving surgical removal and discuss prognosis .

eight members with schwannomatosis from four consecutive generations of a family included in this study were selected according to the following criteria : age : > 30 years ; two or more nonintradermal schwannomas , at least one with histological confirmation ; no evidence of vestibular tumor on high - quality magnetic resonance imaging scan ; and no known constitutional nf2 mutation . the ages at initial symptoms of body pains and numbness were ~3749 years for the disease - affected members . among five individuals with spinal imaging , the spinal schwannoma manifested as intradural , extramedullary nodules in areas of the lumbar , thoracic , and cervical spine . genomic dna ( 3 g ) from peripheral blood mononuclear cells was used for ( i ) the exon capture using the sureselect human all exon kit ( agilent technologies , santa clara , ca ) and ( ii ) sequencing and cluster generation using the illumina genome analyzer iix ( illumina , san diego , ca ) , as we previously reported . single - base substitutions were detected based on minimum total coverage of 10 and minimum variant coverage of 3 . small insertions / deletions and the log2 copy number variation were calculated for tumor versus normal tissue and were median centered , as we previously reported . the saccharomyces cerevisiae coq6-null mutant w303g63 ( mat ade2 - 1 , his 3 - 1,15 leu2 - 3,112 trp1 - 1 ura3 - 1 coq6:his3 ) , yeast coq6 gene expression plasmid , and a low - copy - number ( pqm ) or a high - copy - number ( prcm ) yeast expression plasmid containing the human coq6 open reading frame with an in - frame amino terminal yeast mitochondrial leader sequence ( as positive control for yeast rescuing studies ) were generous gifts of dr catherine f. clarke ( university of california , los angeles ) . the d208h mutation of human coq6 was introduced by site - directed mutagenesis ( stratagene , la jolla , ca ) . the s. cerevisiae coq6-null mutants were unable to survive in media containing a nonfermentable carbon source unless a functional coq6 gene was introduced into them , and the yeast complementation experiment was performed as previously reported . the coq10 level in cells was measured using a human coq10 elisa kit , and this level was converted to nanograms per microgram of total cellular protein . mitochondrial membrane potential ( m ) was measured using the standard mitochondrial - specific dual - fluorescence probe jc-1 ( 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide ) , as previously reported . rat coq6 gene specific small interfering rna and scrambled small interfering rna were transfected into rat schwann cells to decrease coq6 , and then production of intracellular reactive oxygen species ( ros ) in rat schwann cells was also monitored by the fluorescence emission of 2,7-dichlorofluorescein at 72 hours posttransfection . hemagglutinin - tagged human wild - type coq6 and d208h coq6 open reading frames were cloned into the pcdna3.1 ( + ) mammalian expression plasmid and introduced into human fibroblast imr90 cells to assess rescue of coq6 knockdown by small interfering rna . eight members with schwannomatosis from four consecutive generations of a family included in this study were selected according to the following criteria : age : > 30 years ; two or more nonintradermal schwannomas , at least one with histological confirmation ; no evidence of vestibular tumor on high - quality magnetic resonance imaging scan ; and no known constitutional nf2 mutation . the ages at initial symptoms of body pains and numbness were ~3749 years for the disease - affected members . among five individuals with spinal imaging , the spinal schwannoma manifested as intradural , extramedullary nodules in areas of the lumbar , thoracic , and cervical spine . genomic dna ( 3 g ) from peripheral blood mononuclear cells was used for ( i ) the exon capture using the sureselect human all exon kit ( agilent technologies , santa clara , ca ) and ( ii ) sequencing and cluster generation using the illumina genome analyzer iix ( illumina , san diego , ca ) , as we previously reported . single - base substitutions were detected based on minimum total coverage of 10 and minimum variant coverage of 3 . small insertions / deletions and the log2 copy number variation were calculated for tumor versus normal tissue and were median centered , as we previously reported . the saccharomyces cerevisiae coq6-null mutant w303g63 ( mat ade2 - 1 , his 3 - 1,15 leu2 - 3,112 trp1 - 1 ura3 - 1 coq6:his3 ) , yeast coq6 gene expression plasmid , and a low - copy - number ( pqm ) or a high - copy - number ( prcm ) yeast expression plasmid containing the human coq6 open reading frame with an in - frame amino terminal yeast mitochondrial leader sequence ( as positive control for yeast rescuing studies ) were generous gifts of dr catherine f. clarke ( university of california , los angeles ) . the d208h mutation of human coq6 was introduced by site - directed mutagenesis ( stratagene , la jolla , ca ) . the s. cerevisiae coq6-null mutants were unable to survive in media containing a nonfermentable carbon source unless a functional coq6 gene was introduced into them , and the yeast complementation experiment was performed as previously reported . the coq10 level in cells was measured using a human coq10 elisa kit , and this level was converted to nanograms per microgram of total cellular protein . mitochondrial membrane potential ( m ) was measured using the standard mitochondrial - specific dual - fluorescence probe jc-1 ( 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide ) , as previously reported . rat coq6 gene specific small interfering rna and scrambled small interfering rna were transfected into rat schwann cells to decrease coq6 , and then production of intracellular reactive oxygen species ( ros ) in rat schwann cells was also monitored by the fluorescence emission of 2,7-dichlorofluorescein at 72 hours posttransfection . hemagglutinin - tagged human wild - type coq6 and d208h coq6 open reading frames were cloned into the pcdna3.1 ( + ) mammalian expression plasmid and introduced into human fibroblast imr90 cells to assess rescue of coq6 knockdown by small interfering rna . the disease - affected members of the family were a father and his daughter in the first and second generations , respectively , and five of eight siblings in the third generation ( named as s1s8 ) ; in addition , one member of the fourth generation has been diagnosed with schwannomatosis thus far ( figure 1a ) . the schwannomatosis tissues of s2 and s7 displayed similar morphological features under hematoxylin - and - eosin staining ( upper panel in figure 1b ) and positive staining for s-100 protein , clearly indicating their schwann cell origin ( lower panel in figure 1b ) . constitutional mutations in smarcb1 , lztr1 , nf1 , and nf2 genes including point mutations , intragenic insertions / deletions , and gene duplication were not found in the family by whole - genome sequencing analysis . consistent with this observation , genetic abnormalities of smarcb1 , nf1 , and nf2 in the schwannomatosis tissues of s2 and s7 were not discovered by fluorescence in situ hybridization analysis ( supplementary figure s1a c online ) and sanger sequencing . immunohistochemical analysis further showed that smarcb1 ( supplementary figure s2a online ) , nf1 ( supplementary figure s2b online ) , and nf2 ( supplementary figure s2c online ) proteins were normally expressed in these tissues . the distribution of schwannomatosis - affected members in the family indicated an autosomal dominant trait . to explore the genetic differences between the affected and unaffected members , we performed whole - genome sequencing on the genomic dna samples of s4 ( schwannomatosis - affected ) and s5 ( normal ) . comparative analysis of whole - genome sequencing showed that there were no deleted / amplified dna regions in s4 compared with the dna of s5 ( supplementary figure s3 online ) . no genetic abnormalities in the smarcb1 , lztr1 , nf1 , and nf2 genes were further identified by whole - genome sequencing analysis ( supplementary table s2 online ) . we further performed whole - exome sequencing on the genomic dna samples of s2 , s3 , and s7 ( supplementary figure s4 online ) . twelve shared heterozygous variants , including nine missense variants ( supplementary table s3 online ) , were identified by these methods ( supplementary figure s4 online ) . variants of hrnr and gstt2 proteins were assessed as benign by polyphen-2 ( polymorphism phenotyping software , version 2 ) . the de novo heterozygous variants of mypn , coq6 , ckmt1a , cyp11a1 , duox1 , and triobp genes were commonly found in all affected members . on the basis of intensive literature review and mutation prediction of these genetic variants , we focused on a heterozygous mutation ( p.asp208his/d208h ; c.622g > c ) , reference sequence nm_182476.2 ) of the coq6 gene , whose function is essential but not redundant . sanger sequencing analysis showed that the d208h allele was found in the disease - affected but not in normal family members ( figure 1c ) . the messenger rna transcript of the d208h coq6 allele was found in the schwannomatosis tissues of s2 and s7 by reverse transcription polymerase chain reaction ( figure 1d ) . in addition to the heterozygous d208h mutation , molecular analysis did not discover other coq6 genetic variants in these schwannomatosis tissues . coq6 monooxygenase is evolutionarily conserved , and the d208 residue is located at the highly conserved flavin adenine dinucleotide ( fad)-binding domain . the missense mutation altered an amino acid residue that is uniformly conserved from escherichia coli to homo sapiens ( humans ) ( figure 1e , upper panel ) . structural modeling of the fad - binding domain of human coq6 using composer in sybyl ( tripos , st louis , mo ) indicated that the h208 residue would clash with the residues s212 and v214 in the fad - binding domain ( figure 1e , lower panel ) , indicating that h208 may negatively affect coq6 function by interfering with its binding to fad . as shown in figure 2a ( left panel ) , all the transformed coq6-null yeast mutants grew normally in sd - ura selective media ( synthetic defined media with uracil dropout , in which glucose acts as fermentable carbon source ) . the yeast coq6 gene ( prs1-yq6 , positive control ) and wild - type human coq6 ( hwt ) gene , but not the d208h coq6 mutant ( d208h - q6 ) , in both high - copy - number ( prcm ) and low - copy - number ( pqm ) plasmids ( the empty vectors were negative controls ) rescued the growth of coq6-null yeasts plated on ypg media ( yeast extract / peptone / glycerol media , in which glycerol acts as a nonfermentable carbon source ) , which indicated that the d208h coq6 mutant lacked complementation in coq6-deficient yeast mutants . furthermore , we also verified that knockdown of coq6 dramatically decreased coq10 production in human fetal lung fibroblast imr90 cells over a period of 72 hours ( figure 2b ) . knockdown of coq6 in imr90 cells dramatically decreased mitochondrial membrane potential , which may cause cell death by energy depletion , and concurrently increased ros levels over 48 hours ( supplementary figure s5a , b online ) . furthermore , we showed that only reconstitution of wild - type human coq6 , not the d208h coq6 mutant , rescued the imr90 cells after knockdown of coq6 ( figure 2c ) . moreover , we also modeled the haploinsufficiency of coq6 by knockdown of coq6 gene in rat schwann cells and found that ros production increased in a dosage - dependent manner ( figure 2d ) , which might suggest that haploinsufficiency of coq6 may cause chronic ros overproduction in schwann cells . the disease - affected members of the family were a father and his daughter in the first and second generations , respectively , and five of eight siblings in the third generation ( named as s1s8 ) ; in addition , one member of the fourth generation has been diagnosed with schwannomatosis thus far ( figure 1a ) . the schwannomatosis tissues of s2 and s7 displayed similar morphological features under hematoxylin - and - eosin staining ( upper panel in figure 1b ) and positive staining for s-100 protein , clearly indicating their schwann cell origin ( lower panel in figure 1b ) . constitutional mutations in smarcb1 , lztr1 , nf1 , and nf2 genes including point mutations , intragenic insertions / deletions , and gene duplication were not found in the family by whole - genome sequencing analysis . consistent with this observation , genetic abnormalities of smarcb1 , nf1 , and nf2 in the schwannomatosis tissues of s2 and s7 were not discovered by fluorescence in situ hybridization analysis ( supplementary figure s1a c online ) and sanger sequencing . immunohistochemical analysis further showed that smarcb1 ( supplementary figure s2a online ) , nf1 ( supplementary figure s2b online ) , and nf2 ( supplementary figure s2c online ) proteins were normally expressed in these tissues . the distribution of schwannomatosis - affected members in the family indicated an autosomal dominant trait . to explore the genetic differences between the affected and unaffected members , we performed whole - genome sequencing on the genomic dna samples of s4 ( schwannomatosis - affected ) and s5 ( normal ) . comparative analysis of whole - genome sequencing showed that there were no deleted / amplified dna regions in s4 compared with the dna of s5 ( supplementary figure s3 online ) . no genetic abnormalities in the smarcb1 , lztr1 , nf1 , and nf2 genes were further identified by whole - genome sequencing analysis ( supplementary table s2 online ) . we further performed whole - exome sequencing on the genomic dna samples of s2 , s3 , and s7 ( supplementary figure s4 online ) . twelve shared heterozygous variants , including nine missense variants ( supplementary table s3 online ) , were identified by these methods ( supplementary figure s4 online ) . variants of hrnr and gstt2 proteins were assessed as benign by polyphen-2 ( polymorphism phenotyping software , version 2 ) . the de novo heterozygous variants of mypn , coq6 , ckmt1a , cyp11a1 , duox1 , and triobp genes were commonly found in all affected members . on the basis of intensive literature review and mutation prediction of these genetic variants , we focused on a heterozygous mutation ( p.asp208his/d208h ; c.622g > c ) , reference sequence nm_182476.2 ) of the coq6 gene , whose function is essential but not redundant . sanger sequencing analysis showed that the d208h allele was found in the disease - affected but not in normal family members ( figure 1c ) . the messenger rna transcript of the d208h coq6 allele was found in the schwannomatosis tissues of s2 and s7 by reverse transcription polymerase chain reaction ( figure 1d ) . in addition to the heterozygous d208h mutation , molecular analysis did not discover other coq6 genetic variants in these schwannomatosis tissues . coq6 monooxygenase is evolutionarily conserved , and the d208 residue is located at the highly conserved flavin adenine dinucleotide ( fad)-binding domain . the missense mutation altered an amino acid residue that is uniformly conserved from escherichia coli to homo sapiens ( humans ) ( figure 1e , upper panel ) . structural modeling of the fad - binding domain of human coq6 using composer in sybyl ( tripos , st louis , mo ) indicated that the h208 residue would clash with the residues s212 and v214 in the fad - binding domain ( figure 1e , lower panel ) , indicating that h208 may negatively affect coq6 function by interfering with its binding to fad . as shown in figure 2a ( left panel ) , all the transformed coq6-null yeast mutants grew normally in sd - ura selective media ( synthetic defined media with uracil dropout , in which glucose acts as fermentable carbon source ) . the yeast coq6 gene ( prs1-yq6 , positive control ) and wild - type human coq6 ( hwt ) gene , but not the d208h coq6 mutant ( d208h - q6 ) , in both high - copy - number ( prcm ) and low - copy - number ( pqm ) plasmids ( the empty vectors were negative controls ) rescued the growth of coq6-null yeasts plated on ypg media ( yeast extract / peptone / glycerol media , in which glycerol acts as a nonfermentable carbon source ) , which indicated that the d208h coq6 mutant lacked complementation in coq6-deficient yeast mutants . furthermore , we also verified that knockdown of coq6 dramatically decreased coq10 production in human fetal lung fibroblast imr90 cells over a period of 72 hours ( figure 2b ) . knockdown of coq6 in imr90 cells dramatically decreased mitochondrial membrane potential , which may cause cell death by energy depletion , and concurrently increased ros levels over 48 hours ( supplementary figure s5a , b online ) . furthermore , we showed that only reconstitution of wild - type human coq6 , not the d208h coq6 mutant , rescued the imr90 cells after knockdown of coq6 ( figure 2c ) . moreover , we also modeled the haploinsufficiency of coq6 by knockdown of coq6 gene in rat schwann cells and found that ros production increased in a dosage - dependent manner ( figure 2d ) , which might suggest that haploinsufficiency of coq6 may cause chronic ros overproduction in schwann cells . coq10 is an electron carrier in the mitochondrial respiratory chain and is also a lipid - soluble antioxidant implicated in protecting cells from damage by ros . a previous study showed that severe coq10 deficiency ( < 30% ) caused a marked defect in bioenergetics , with decreased adenosine triphosphate production and sometimes decreased cell growth , but no increase in ros or oxidative stress induced death . by contrast , intermediate decreases in coq10 ( 3050% ) caused mild defects in bioenergetics , with significant increases in ros and oxidative stress induced cell death . our knockdown of coq6 in imr90 cells also resulted in a similar reduction of cellular coq10 and mitochondrial membrane potential . we also identified that knockdown of coq6 impaired mitochondrial function and increased ros overproduction in rat schwann cells , with a positive correlation between decreased coq6 protein and ros production . elevated levels of ros and decreased levels of ros scavengers and antioxidant enzymes are associated with various human diseases including cancers . for example , increased ros production caused by mitochondrial dna mutations has been linked to tumor progression in prostate cancer models . abnormally low plasma levels of coq10 have been found in a number of cancer types , including cervical cancer and melanoma . decreased levels of coq10 have been detected in human breast cancer tissues . however , whether deficiency of coq10 predisposes individuals to certain cancers is not known . similarly , the roles of coq10 biosynthesis gene mutations have not been appreciated in cancers . the association is only now becoming apparent with the identification of coq2 gene ( one of the coq10 biosynthesis enzymes ) mutations in human colon and rectal cancers , ovarian carcinoma , and glioblastoma multiforme by cancer genomic studies . in this study , we have identified a heterozygous loss - of - function coq6 missense mutation in familial schwannomatosis . a critical issue related to implication of the genetic alteration in the familial disease is whether the heterozygous loss - of - function coq6 displays haploinsufficiency . a large - scale screening study for genes in yeast displaying haploinsufficiency found that haploinsufficiency of coq6 resulted in a mild reduction of fitness in a medium containing glucose . a recent study demonstrated that both homozygous and compound heterozygous loss - of - function mutations in the human coq6 gene caused early - onset steroid - resistant nephritic syndrome with sensorineural deafness , which progressed to end - stage renal failure by a median age of < 2 years , and two single heterozygous nonsense mutations were also identified in two families with cyclosporin a therefore , it may be assumed that the coq6 haploinsufficiency may be conditional and tissue / cell specific . in the family under study , none of the members harboring the mutated allele had nephritic syndrome , but they did have schwannomatosis , indicating diversity and heterogeneity of clinical phenotypes caused by defects of the same gene . germline abnormalities associated with cancer may be detected in every cell in the body or only in the tumor cells . interestingly , despite the presence of a constitutional genetic abnormality that might affect growth regulatory pathways in all cells , people are generally predisposed to only certain tumor types . in this particular family , we consider that the d208h coq6 allele may lead to a chronic or conditional haploinsufficiency of coq6 in a cell / tissue - specific manner , causing chronic ros overproduction in schwann cells through an unknown mechanism , thus predisposing the family members to familial schwannomatosis . in summary , although the exact oncogenetic mechanism of the loss - of - function coq6 gene in the disease remains a challenging question to be elucidated , we have , for the first time , suggested an association of the defect in one of the coq10 biosynthesis genes , coq6 , with familial schwannomatosis . the mutated coq6 allele may lead to coq10 deficiency and chronic overproduction of ros in schwann cells , which may predispose individuals to the disease . emergence of bacterial resistance to many different antibiotics is considered as a great concern in human health . pseudomonas aeruginosa ( pa ) has been recognized as one of the significant pathogens of nosocomial infections ( 1 ) . key mechanism of antibiotic resistance in p. aeruginosa is the expulsion of antibiotics through multidrug resistance ( mdr ) efflux systems belonging to the resistance - nodulation - division ( rnd ) family ( 2 ) . mexab - oprm , mexcd - oprj , mexef - oprn , mexxy , mexjk and mexvw contribute the most significantly to antibiotic resistance ( 3 ) and play an important role in intrinsic and acquired multidrug resistance ( 2 ) . resistance of p. aeruginosa to multiple antibiotics is largely attributable to expression of the mexab - oprm efflux pump ( 4 ) . among all the rnd pumps of pa , mex - ab - oprm was the first efflux pump found to target multiple classes of antibiotics including -lactam ( carboxypenicillins , aztreonam , extended - spectrum cephalosporins , penems , the carbapenems such as meropenem and panipenem except imipenem and biapenem ) ; fluoroquinolones , tetracyclines , chloramphenicol , macrolides , novobiocin , trimethoprim and sulfonamides ( 5 , 6 ) . the mexab - oprm efflux pump belongs to the superfamily of ribonucleoproteins and consists of an inner membrane ( mexb ) , a periplasmic membrane fusion protein ( mexa ) and a channel - forming outer membrane protein , oprm ( 7 ) . in this study , we investigated the role of mex - ab - oprm efflux pump . transcription level of efflux pump genes mexa , mexb , oprm , mexr and ampc were analyzed using real - time pcr . p. aeruginosa strains were isolated from patients of children s hospital medical center during 6 months ( march and august 2012 ) . all bacterial isolates were identified at microbiology lab using standard biochemical identification methods ( 8) . a total of 45 samples were collected from various clinical specimens such as urine ( n=21 ) , exudates ( n=11 ) , eye ( n=2 ) , ear ( n=2 ) , csf ( n=2 ) , blood ( n=2 ) , trachea ( n=2 ) and lung secretions ( n=3 ) . p.aeruginosa pao1 strain which has entirely sequenced genome was used as the reference wild type strain throughout the study . antimicrobial susceptibility tests were performed using disk diffusion and minimum inhibitory concentration ( mic ) methods . antibiotics used in this study were chosen randomly from different antibiotic classes that were used mostly in this hospital . antibiotics used in the disk diffusion method were cephalothin ( 30 g ) , cefepime ( 30 g ) , ceftazidime ( 30 g ) , ciprofloxacin ( 5 g ) ( flouroquionolones ) , meropenem ( 10 g ) , imipenem ( 10 g ) ( carbapenems ) , piperacillin / tazobactam ( 10/100 g ) ( -lactams ) , gentamicin ( 10 g ) and amikacin ( 30 g ) ( aminoglycosides ) . antibiotics used in this study were cefuroxime , ceftazidim , cefazolin , ceftriaxone ( cephems ) , meropenem ( carbapenems ) , amikacin , tobramycin ( aminoglycosides ) , aztreonam ( monobactam ) , ampicillin , piperacillin ( penicillins ) , colistin ( lipopeptides ) , gatifloxacin , nalidixic acid ( flouroquionolones ) , piperacillin - tazobactam , tazobactam ( -lactams ) ( mast , uk ) . total rna was extracted using rna extraction kit ( fermentas , lithuania ) , and converted into cdna using the cdna synthesis kit ( fermentas , lithuania ) according to the manufacturer s instruction and the quality and purity of the rna obtained was evaluated using spectrophotometer . the final optimized pcr reaction consisted of 1 l of each primer ( 10pmol ) ( table 1 ) , 0.5 l dntp ( 10 mm ) , 0.5 l mgcl ( 100 mm ) , 0.2 l ( 1 unit ) taq dna polymerase ( metabion , germany ) , 2.5 l pcr buffer ( 10x ) , and 0.5 l of dna template ( 100g / ml ) in total volume of 25 l with double distilled water . the cycling program was adjusted as follows : initial denaturation at 94 c for 5 min followed by 30 cycles of 94 c for 45 sec , 5072 c ( 45 sec ) , 72 c ( 1 min ) and a final extension at 72 c for 10 min ( 9 ) . primers used in pcr and real - time pcr real - time quantification of cdna was carried out on an abi step one detection system ( applied biosystems , uk ) using the sybr green pcr master mix . real - time pcr was used to investigate the expression level of each gene in mexab - oprm efflux pump to expression each of these genes of separately measured by relative quantitation real - time pcr . the optimized reaction consisted of master mix ( 10x ) , 1 l of each primer ( 10 pmol ) , and 0.5 l of template dna ( 100 g / ml ) in a total volume of 20 l p. aeruginosa strain pao1 was used as a standard strain for normalization of relative mrna levels . ampc gene was considered as a housekeeping gene and all gene expressions were compared with ampc gene expression . the assay was performed three times for each sample and the mean of three obtained quantities was considered as quantity . cycle of threshold ( c t ) was considered as the average threshold cycle number from three independent experiments . the real - time pcr apparatus was programmed as follows : initial denaturation at 95 c ( 15 min ) followed by 40 cycles of 95 c ( 15 sec ) , 61 c ( 15 sec ) , 71 c ( 20 sec ) and melt curve at 61 c ( 1 min ) and 95 c for 15 sec . to obtain reproducibility of the reaction ampc gene was considered as a housekeeping gene and the results were compared with gene expression in a susceptible pa reference strain . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification had occurred , melting curves of each amplicon were assessed p. aeruginosa strain pao1 was used as a standard strain for normalization of relative mrna levels . real - time pcr efficiencies were acquired by amplification of a standardized dilution series of the template cdna and were determined for each gene as the slope of a linear regression model . pcr efficiency was determined by measuring the ct ct to a specific threshold for a serial dilution of cdna . the corresponding real - time pcr efficiencies were then calculated according to the equation : e = ( 101 ) 100 ( 1012 ) . p. aeruginosa strains were isolated from patients of children s hospital medical center during 6 months ( march and august 2012 ) . all bacterial isolates were identified at microbiology lab using standard biochemical identification methods ( 8) . a total of 45 samples were collected from various clinical specimens such as urine ( n=21 ) , exudates ( n=11 ) , eye ( n=2 ) , ear ( n=2 ) , csf ( n=2 ) , blood ( n=2 ) , trachea ( n=2 ) and lung secretions ( n=3 ) . p.aeruginosa pao1 strain which has entirely sequenced genome was used as the reference wild type strain throughout the study . antimicrobial susceptibility tests were performed using disk diffusion and minimum inhibitory concentration ( mic ) methods . antibiotics used in this study were chosen randomly from different antibiotic classes that were used mostly in this hospital . antibiotics used in the disk diffusion method were cephalothin ( 30 g ) , cefepime ( 30 g ) , ceftazidime ( 30 g ) , ciprofloxacin ( 5 g ) ( flouroquionolones ) , meropenem ( 10 g ) , imipenem ( 10 g ) ( carbapenems ) , piperacillin / tazobactam ( 10/100 g ) ( -lactams ) , gentamicin ( 10 g ) and amikacin ( 30 g ) ( aminoglycosides ) . antibiotics used in this study were cefuroxime , ceftazidim , cefazolin , ceftriaxone ( cephems ) , meropenem ( carbapenems ) , amikacin , tobramycin ( aminoglycosides ) , aztreonam ( monobactam ) , ampicillin , piperacillin ( penicillins ) , colistin ( lipopeptides ) , gatifloxacin , nalidixic acid ( flouroquionolones ) , piperacillin - tazobactam , tazobactam ( -lactams ) ( mast , uk ) . total rna was extracted using rna extraction kit ( fermentas , lithuania ) , and converted into cdna using the cdna synthesis kit ( fermentas , lithuania ) according to the manufacturer s instruction and the quality and purity of the rna obtained was evaluated using spectrophotometer . the final optimized pcr reaction consisted of 1 l of each primer ( 10pmol ) ( table 1 ) , 0.5 l dntp ( 10 mm ) , 0.5 l mgcl ( 100 mm ) , 0.2 l ( 1 unit ) taq dna polymerase ( metabion , germany ) , 2.5 l pcr buffer ( 10x ) , and 0.5 l of dna template ( 100g / ml ) in total volume of 25 l with double distilled water . the cycling program was adjusted as follows : initial denaturation at 94 c for 5 min followed by 30 cycles of 94 c for 45 sec , 5072 c ( 45 sec ) , 72 c ( 1 min ) and a final extension at 72 c for 10 min ( 9 ) . real - time quantification of cdna was carried out on an abi step one detection system ( applied biosystems , uk ) using the sybr green pcr master mix . real - time pcr was used to investigate the expression level of each gene in mexab - oprm efflux pump to expression each of these genes of separately measured by relative quantitation real - time pcr . the optimized reaction consisted of master mix ( 10x ) , 1 l of each primer ( 10 pmol ) , and 0.5 l of template dna ( 100 g / ml ) in a total volume of 20 l p. aeruginosa strain pao1 was used as a standard strain for normalization of relative mrna levels . ampc gene was considered as a housekeeping gene and all gene expressions were compared with ampc gene expression . the assay was performed three times for each sample and the mean of three obtained quantities was considered as quantity . cycle of threshold ( c t ) was considered as the average threshold cycle number from three independent experiments . the real - time pcr apparatus was programmed as follows : initial denaturation at 95 c ( 15 min ) followed by 40 cycles of 95 c ( 15 sec ) , 61 c ( 15 sec ) , 71 c ( 20 sec ) and melt curve at 61 c ( 1 min ) and 95 c for 15 sec . to obtain reproducibility of the reaction ampc gene was considered as a housekeeping gene and the results were compared with gene expression in a susceptible pa reference strain . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification had occurred , melting curves of each amplicon were assessed p. aeruginosa strain pao1 was used as a standard strain for normalization of relative mrna levels . real - time pcr efficiencies were acquired by amplification of a standardized dilution series of the template cdna and were determined for each gene as the slope of a linear regression model . pcr efficiency was determined by measuring the ct ct to a specific threshold for a serial dilution of cdna . the corresponding real - time pcr efficiencies were then calculated according to the equation : e = ( 101 ) 100 ( 1012 ) . totally , 45 strains of p. aeruginosa were isolated from patients aged 2 months to 12 years who were referred to children s medical center hospital , tehran , iran . the majority of isolates were collected from patients hospitalized in picu ( n=18 , 39% ) , nicu ( n=5 , 11% ) followed by emergency ward ( n=3 , 7% ) , nephrology ( n=3 , 7% ) , surgery ( n=5 , 11% ) , neurology ( n=3 , 7% ) , urology ( n=5 , 11% ) and infectious ward ( n=3 , 7% ) . the most antibiotic resistance based on clsi reference guidelines for disk diffusion method was detected for cephalothin ( n=41 , 92% ) whereas mics should the following frequencies for antibiotic resistance : cefuroxime ( n=41 , 91% ) , ceftazidime ( n=42 , 93% ) , amikacin ( n=38 , 84.5% ) , cefazolin ( n=40 , 89% ) , aztreonam ( n=42 , 93% ) , piperacillin ( n=39 , 86.5% ) , tazobactam ( n=41 , 91% ) and piperacillin - tazobactam ( n=42 , 93% ) . antibiotics such as colistin ( 15% ) , ceftriaxone ( 33% ) and tobramycin ( 22% ) showed the highest susceptibility rates against the isolates ( table 2 ) . pattern of determined antibiotic resistance among p. aeruginosa strains using disk diffusion and mic methods the best annealing temperature was obtained at 61 c . the results of real - time pcr for the genes of mexab - oprm efflux pump have been shown in table 3 . according to the results of antibiotic resistance via disk diffusion method , overexpression of mexab - oprm genes was associated to the resistance towards cephalosporin while via mic method overexpression of mexab - oprm was seen in those showing resistance towards special antibiotics especially fluoroquinolones , cephalosporin and beta lactams . the expression of mexab - oprm efflux pump genes in p. aeruginosa strains isolated from children the reproducibility of the expression levels of each gene was measured three times . p. aeruginosa clinical isolates demonstrated increased level of mexa ( 2 folds ) , mexb ( from 2.2 to 12.0 folds ) , oprm ( 2 folds ) and ampc ( 10 folds ) at transcriptional mrna level , respectively . confirmation of specific amplifications during real - time pcr on mexab - oprm genes of efflux pump among 45 resistant isolates , overexpression of mexa gene was observed in 25 isolates ( 55.5% ) , mexb in 24 isolates ( 53.3% ) and oprm in 16 isolates ( 35.5% ) . in 28 isolates ( 62% ) , overexpression was observed in one of the three genes of mexab - oprm efflux pump . the ratio values obtained for each of the four genes in samples showed that the expression levels of mexa gene ( n=25 , 89% ) was between 1.8 and 11.7 ( mean= 6.7 ) , mexb ( n=24 , 85% ) between 1.3 and 14 ( mean= 7.6 ) and oprm gene ( n=16 , 57% ) between 1 and 9 ( mean= 5 ) . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification ( fig . totally , 45 strains of p. aeruginosa were isolated from patients aged 2 months to 12 years who were referred to children s medical center hospital , tehran , iran . the majority of isolates were collected from patients hospitalized in picu ( n=18 , 39% ) , nicu ( n=5 , 11% ) followed by emergency ward ( n=3 , 7% ) , nephrology ( n=3 , 7% ) , surgery ( n=5 , 11% ) , neurology ( n=3 , 7% ) , urology ( n=5 , 11% ) and infectious ward ( n=3 , 7% ) . the most antibiotic resistance based on clsi reference guidelines for disk diffusion method was detected for cephalothin ( n=41 , 92% ) whereas mics should the following frequencies for antibiotic resistance : cefuroxime ( n=41 , 91% ) , ceftazidime ( n=42 , 93% ) , amikacin ( n=38 , 84.5% ) , cefazolin ( n=40 , 89% ) , aztreonam ( n=42 , 93% ) , piperacillin ( n=39 , 86.5% ) , tazobactam ( n=41 , 91% ) and piperacillin - tazobactam ( n=42 , 93% ) . antibiotics such as colistin ( 15% ) , ceftriaxone ( 33% ) and tobramycin ( 22% ) showed the highest susceptibility rates against the isolates ( table 2 ) . pattern of determined antibiotic resistance among p. aeruginosa strains using disk diffusion and mic methods the results of real - time pcr for the genes of mexab - oprm efflux pump have been shown in table 3 . according to the results of antibiotic resistance via disk diffusion method , overexpression of mexab - oprm genes was associated to the resistance towards cephalosporin while via mic method overexpression of mexab - oprm was seen in those showing resistance towards special antibiotics especially fluoroquinolones , cephalosporin and beta lactams . the expression of mexab - oprm efflux pump genes in p. aeruginosa strains isolated from children the reproducibility of the expression levels of each gene was measured three times . p. aeruginosa clinical isolates demonstrated increased level of mexa ( 2 folds ) , mexb ( from 2.2 to 12.0 folds ) , oprm ( 2 folds ) and ampc ( 10 folds ) at transcriptional mrna level , respectively . confirmation of specific amplifications during real - time pcr on mexab - oprm genes of efflux pump among 45 resistant isolates , overexpression of mexa gene was observed in 25 isolates ( 55.5% ) , mexb in 24 isolates ( 53.3% ) and oprm in 16 isolates ( 35.5% ) . in 28 isolates ( 62% ) , overexpression was observed in one of the three genes of mexab - oprm efflux pump . the ratio values obtained for each of the four genes in samples showed that the expression levels of mexa gene ( n=25 , 89% ) was between 1.8 and 11.7 ( mean= 6.7 ) , mexb ( n=24 , 85% ) between 1.3 and 14 ( mean= 7.6 ) and oprm gene ( n=16 , 57% ) between 1 and 9 ( mean= 5 ) . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification ( fig . p. aeruginosa is one of the most important causes of nosocomial infections due to the presence of various resistant elements ( 11 ) . during last decades , the emergency of multidrug resistant p. aeruginosa has been observed worldwide . in this study , 2835% of studied p. aeruginosa isolates were resistant to carbapenems which was in accordance with previous studies ( 13 , 14 ) . high frequency of cephalosporin resistance was also observed among p. aeruginosa isolates similar to the other reports ( 14 , 15 ) . among all isolates , resistance to piperacillin / tazobactam was low ( 2627% ) which was in agreement with our previous report ( 13 ) and was higher than ghazi et al . report ( 16 ) . in this study colistin showed 15% resistance which can be suggested as an effective antibiotic for treatment of pa infections . this finding is similar to the results of study performed by alekshun et al . in us ( 17 ) . at present , the efflux pump has been recognized as one of the significant complexes involved in resistance to most of the classes of antibiotics ( 11 , 18 , 19 ) . it has been reported that the prevalence of efflux pump overexpression in clinical p. aeruginosa strains of ranged from 1475% ( 20 , 21 ) . there are rare reports on prevalence of efflux pump overexpression in our country ( 22 , 23 ) and there is no investigation on pa strains isolated from children . in the present study the increased expression level of mexab - oprm genes of efflux pump simultaneously was 25% which was relatively more than dumas et al . ( 24 ) . in this study , 28 of 45 patients ( 62% ) showed an increased expression level of efflux pumps mex - ab - oprm genes that was similar to the studies reported more than 50% overexpression level of these genes ( 2427 ) . in arabestani study all the isolates ( n=31 ; 100% ) showed overexpression of efflux pump mexab - oprm genes ( 23 ) . according to aghazadeh et al . report , overexpression of mexa was 74% among the isolates ( 28 ) . since the concomitant overproduction of two mex pumps might have additive effects on being resistant to antibiotics ( 29 ) evaluating the co - expression of multi - component efflux pumps other than mexab - oprm is recommended . development of novel antibiotics that can bypass the effects of efflux pumps is still a challenging task . further studies on involved mechanisms and structure - function association of bacterial efflux systems as well as the interactions between the pumps and other resistance mechanisms are highly recommended .

purpose : schwannomatosis , a subtype of neurofibromatosis , is characterized by multiple benign , nonvestibular , nonintradermal schwannomas . although the tumor suppressor smarcb1 gene has been frequently identified as the underlying genetic cause of half of familial and ~10% of sporadic schwannomatosis , for most other cases , further causative genes remain to be discovered . herein , we characterize the genome of a schwannomatosis family without constitutional inactivation of the smarcb1 gene to explore novel genomic alterations predisposing individuals to the familial disease.methods:we performed whole - genome / exome sequencing on genomic dna of both schwannomatosis - affected and normal members of the family.results:we identified a novel missense mutation ( p.asp208his ; c.622g > c ) in the coenzyme q10 ( coq10 ) biosynthesis monooxygenase 6 gene ( coq6 ) in schwannomatosis - affected members . the deleterious effects of the coq6 mutations were validated by their lack of complementation in a coq6-deficient yeast mutant . our study further indicated that the resultant haploinsufficiency of coq6 might lead to coq10 deficiency and chronic overproduction of reactive oxygen species in schwann cells.conclusion:although the exact oncogenetic mechanisms in this schwannomatosis family remain to be elucidated , our data strongly indicate a probable role of coq6 mutation and coq10 deficiency in the development of familial schwannomatosis . background and objectives : pseudomonas aeruginosa ( pa ) is one of the most important causes of nosocomial infections and has an intrinsic resistance to many antibiotics . among all the resistance - nodulation - division ( rnd ) pumps of p. aeruginosa , mexab - oprm is the first efflux pump found to target multiple classes of antibiotics . this study was aimed to evaluate the expression level of genes expressing mexab - oprm in clinical isolates of p. aeruginosa.materials and methods : in this study , 45 p. aeruginosa strains were isolated from patients admitted to children s medical center hospital , an iranian referral hospital . disk diffusion and minimum inhibitory concentration ( mic ) methods were used for determination of the patterns of resistance to antibiotics . real - time pcr was used to investigate the expression level of genes of mexab - oprm efflux pump.results:among 45 resistant pa isolates , the frequency of genes overexpression was as follows : mexa ( n=25 , 55.5% ) , mexb ( n=24 , 53.3% ) and oprm ( n=16 , 35.5% ) . in addition , in 28 strains ( 62% ) overexpression was observed in one of the studied three genes of mexab - oprm efflux pump.conclusion:in our study 28 isolates ( 62% ) had increased expression level of efflux pumps genes , mexab - oprm . although the efflux pumps play important roles in increasing the resistance towards different antibiotics but the role of other agents and mechanisms in evolution of resistance should not be ignored . since the concomitant overproduction of other mex efflux systems might have additive effects on antibiotic resistance , the co - expressing of a multicomponent efflux pump is recommended . on the other hand , the concomitant overproduction of two mex pumps might have additive effects on resistance to antibiotic . therefore co - expressing of mex efflux systems is recommended .

severe sepsis and septic shock are common causes of mortality and morbidity in an intensive care unit ( icu ) setting . the endotoxin ( lipopolysaccharide [ lps ] ) derived from the outer membranes of gram - negative bacteria is considered a major factor in the pathogenesis of sepsis . the toll - like receptor ( tlr ) family can be found in mammalian cells . endotoxins transduce their signal through the tlr4 transmembrane receptor , and innate immune cascades are initiated , which promote excessive cytokine release and tissue damage . the endotoxin level is associated with clinical outcome and higher activity correlates with greater icu mortality . therapeutic strategies aimed at minimizing or preventing the action of endotoxins are , therefore , attractive . however , the blockage of an endotoxin via binding with monoclonal antibodies has failed to improve outcome in clinical studies . the reduction of endotoxin levels or blockage of endotoxins can potentially interrupt the biological cascade of sepsis . polymyxin b ( pmx ) is an antibiotic agent that has strong gram - negative bactericidal activity and carries very high affinity for endotoxins . intravenous ( iv ) administration of pmx has significant nephrotoxicity and neurotoxicity , which has limited its clinical use . pmx can be immobilized covalently on polystyrene - based carrier fibers which preserve its endotoxin binding capacity without producing toxicity . a pmx immobilized fiber column was shown to improve blood pressure , oxygenation and mortality in patients with severe sepsis . the alteco endotoxin hemoadsorber ( alteco medical ab , lund , sweden ) is a similar device with strong endotoxin - binding capacity . during the treatment , we performed this randomized controlled trial ( rct ) in patients who suffered from septic shock due to intra - abdominal sepsis . we hypothesized that alteco endotoxin hemoadsorption may provide extraclinical benefit in terms of faster organ function improvement and hemodynamic stabilization when compared with conventional treatment . this prospective rct was approved by the institution 's ethics committee and registered with australian new zealand clinical trials registry ( anzctr , actrn12610000892011 ) . the study was conducted in the adult icu of pamela youde nethersole eastern hospital , which is a 2300-bed acute care tertiary hospital that provides comprehensive care , except for cardiothoracic surgery , transplant surgery , and burns . the icu is a 22-bed closed mixed medical - surgical unit with an average admission of 1400 patients / year . we enrolled patients who fulfilled the following inclusion criteria : ( 1 ) age 18 and 85 years old ; ( 2 ) presence of severe sepsis due to intra - abdominal infection where severe sepsis was defined using the american college of chest physicians / society of critical care medicine / european society of intensive care medicine criteria ; ( 3 ) presence of shock with mean arterial pressure ( map ) 65 mmhg ; ( 4 ) requirement of vasopressor support ( noradrenaline 0.2 g / kg / min or equivalent ) ; and ( 5 ) on hydrocortisone 200 - 300 mg iv / day or equivalent to cover potential relative adrenal insufficiency . exclusion criteria of the study were : pregnancy , terminally ill patients with life expectancy 3 months , hypersensitivity to heparin or low molecular heparin or any component of the formulation , known history of heparin - induced thrombocytopenia ; severe thrombocytopenia ( < 50,000/mm ) , uncontrolled active bleeding except when due to disseminated intravascular coagulation , and inclusion in other icu studies . informed consent was obtained from patients directly . for those with impaired consciousness due to underlying illness or the use of sedatives block randomization was performed using a computer generated scheme , and the allocation sequence was concealed in sealed envelopes which were available 24 h a day in the icu . the control group ( cg ) received conventional therapy for septic shock , namely : infective sources control , early appropriate antibiotics , fluid challenge and vasopressor infusion , and lung protected ventilatory strategy based on surviving sepsis campaign guidelines . continuous renal replacement therapy ( crrt ) in the form of citrate - based postdilution continuous venovenous hemofiltration using polysulfone high flux hemofilter ( f 80 , fresenius medical care , germany ) was provided in the presence of acute kidney injury categorized as injury or more based on risk , injury , failure , loss , and end - stage ( rifle ) criteria . treatment group ( tg ) ( endotoxin hemoadsorption group ) received endotoxin hemoadsorption in addition to conventional therapy . a double lumen 12-f hemodialysis catheter ( arrowguard blue plus antimicrobial catheter , arrow international inc . , usa ) was inserted into either the internal jugular or femoral vein for vascular access by the attending intensivists / physicians immediately after randomization . endotoxin hemoadsorption was performed with alteco endotoxin hemoadsorber using ak10 machine ( gambro - hospal , stockholm , sweden ) at a blood flow rate of 120 - 150 ml / h . each patient received two 2-hourly sessions of hemoadsorption 24 h apart . low molecular weight heparin ( lmwh ) was used for anticoagulation at the discretion of the treating physician , with tinzaparin 1000 iu iv as the default dosage . crrt was started in - between two sessions of hemoadsorption and afterwards based on the same starting criteria for cg if necessary . disease severity and prognosis were assessed with acute physiology and chronic health evaluation ( apache ) iv score . clinical parameters and laboratory data were recorded at 0 , 24 , 48 , and 72 h of randomization . dosage of vasopressor was expressed as vasopressor score ( vs ) using the formula : ( dopamine dose 1 ) + ( dobutamine dose 1 ) + ( adrenaline dose 100 ) + ( noradrenaline dose 100 ) + ( phenylephrine dose 100 ) , wherein all doses are expressed as g / kg / min . dose - response relationship between vasopressor and blood pressure was expressed as vasopressor dependency index ( vdi ) and was calculated using the formula : vs / map . primary end point was the change of the sofa score from 0 to 72 h of randomization . secondary end points included changes of vs , vdi , pao2/fio2 ratio , icu length of stay ( los ) , hospital los , and 28-day mortality . sample size was estimated based on previous study findings . with the power of 80% , type i error probability of 0.05 , mean sofa score difference of 2 , standard deviation of 1.5 , the estimated sample size was 20 . univariate analysis was performed using fisher 's exact test for categorical data or mann - whitney u - test for continuous data where appropriate . the analysis was performed by the statistical package for social sciences for windows , version 16.0 ( spss , chicago , il , united states ) . this prospective rct was approved by the institution 's ethics committee and registered with australian new zealand clinical trials registry ( anzctr , actrn12610000892011 ) . the study was conducted in the adult icu of pamela youde nethersole eastern hospital , which is a 2300-bed acute care tertiary hospital that provides comprehensive care , except for cardiothoracic surgery , transplant surgery , and burns . the icu is a 22-bed closed mixed medical - surgical unit with an average admission of 1400 patients / year . we enrolled patients who fulfilled the following inclusion criteria : ( 1 ) age 18 and 85 years old ; ( 2 ) presence of severe sepsis due to intra - abdominal infection where severe sepsis was defined using the american college of chest physicians / society of critical care medicine / european society of intensive care medicine criteria ; ( 3 ) presence of shock with mean arterial pressure ( map ) 65 mmhg ; ( 4 ) requirement of vasopressor support ( noradrenaline 0.2 g / kg / min or equivalent ) ; and ( 5 ) on hydrocortisone 200 - 300 mg iv / day or equivalent to cover potential relative adrenal insufficiency . exclusion criteria of the study were : pregnancy , terminally ill patients with life expectancy 3 months , hypersensitivity to heparin or low molecular heparin or any component of the formulation , known history of heparin - induced thrombocytopenia ; severe thrombocytopenia ( < 50,000/mm ) , uncontrolled active bleeding except when due to disseminated intravascular coagulation , and inclusion in other icu studies . informed consent was obtained from patients directly . for those with impaired consciousness due to underlying illness or the use of sedatives block randomization was performed using a computer generated scheme , and the allocation sequence was concealed in sealed envelopes which were available 24 h a day in the icu . the control group ( cg ) received conventional therapy for septic shock , namely : infective sources control , early appropriate antibiotics , fluid challenge and vasopressor infusion , and lung protected ventilatory strategy based on surviving sepsis campaign guidelines . continuous renal replacement therapy ( crrt ) in the form of citrate - based postdilution continuous venovenous hemofiltration using polysulfone high flux hemofilter ( f 80 , fresenius medical care , germany ) was provided in the presence of acute kidney injury categorized as injury or more based on risk , injury , failure , loss , and end - stage ( rifle ) criteria . treatment group ( tg ) ( endotoxin hemoadsorption group ) received endotoxin hemoadsorption in addition to conventional therapy . a double lumen 12-f hemodialysis catheter ( arrowguard blue plus antimicrobial catheter , arrow international inc . , usa ) was inserted into either the internal jugular or femoral vein for vascular access by the attending intensivists / physicians immediately after randomization . endotoxin hemoadsorption was performed with alteco endotoxin hemoadsorber using ak10 machine ( gambro - hospal , stockholm , sweden ) at a blood flow rate of 120 - 150 ml / h . each patient received two 2-hourly sessions of hemoadsorption 24 h apart . low molecular weight heparin ( lmwh ) was used for anticoagulation at the discretion of the treating physician , with tinzaparin 1000 iu iv as the default dosage . crrt was started in - between two sessions of hemoadsorption and afterwards based on the same starting criteria for cg if necessary . disease severity and prognosis were assessed with acute physiology and chronic health evaluation ( apache ) iv score . clinical parameters and laboratory data were recorded at 0 , 24 , 48 , and 72 h of randomization . dosage of vasopressor was expressed as vasopressor score ( vs ) using the formula : ( dopamine dose 1 ) + ( dobutamine dose 1 ) + ( adrenaline dose 100 ) + ( noradrenaline dose 100 ) + ( phenylephrine dose 100 ) , wherein all doses are expressed as g / kg / min . dose - response relationship between vasopressor and blood pressure was expressed as vasopressor dependency index ( vdi ) and was calculated using the formula : vs / map . primary end point was the change of the sofa score from 0 to 72 h of randomization . secondary end points included changes of vs , vdi , pao2/fio2 ratio , icu length of stay ( los ) , hospital los , and 28-day mortality . sample size was estimated based on previous study findings . with the power of 80% , type i error probability of 0.05 , mean sofa score difference of 2 , standard deviation of 1.5 , the estimated sample size was 20 . univariate analysis was performed using fisher 's exact test for categorical data or mann - whitney u - test for continuous data where appropriate . the analysis was performed by the statistical package for social sciences for windows , version 16.0 ( spss , chicago , il , united states ) . this prospective rct was approved by the institution 's ethics committee and registered with australian new zealand clinical trials registry ( anzctr , actrn12610000892011 ) . the study was conducted in the adult icu of pamela youde nethersole eastern hospital , which is a 2300-bed acute care tertiary hospital that provides comprehensive care , except for cardiothoracic surgery , transplant surgery , and burns . the icu is a 22-bed closed mixed medical - surgical unit with an average admission of 1400 patients / year . we enrolled patients who fulfilled the following inclusion criteria : ( 1 ) age 18 and 85 years old ; ( 2 ) presence of severe sepsis due to intra - abdominal infection where severe sepsis was defined using the american college of chest physicians / society of critical care medicine / european society of intensive care medicine criteria ; ( 3 ) presence of shock with mean arterial pressure ( map ) 65 mmhg ; ( 4 ) requirement of vasopressor support ( noradrenaline 0.2 g / kg / min or equivalent ) ; and ( 5 ) on hydrocortisone 200 - 300 mg iv / day or equivalent to cover potential relative adrenal insufficiency . exclusion criteria of the study were : pregnancy , terminally ill patients with life expectancy 3 months , hypersensitivity to heparin or low molecular heparin or any component of the formulation , known history of heparin - induced thrombocytopenia ; severe thrombocytopenia ( < 50,000/mm ) , uncontrolled active bleeding except when due to disseminated intravascular coagulation , and inclusion in other icu studies . informed consent was obtained from patients directly . for those with impaired consciousness due to underlying illness or the use of sedatives block randomization was performed using a computer generated scheme , and the allocation sequence was concealed in sealed envelopes which were available 24 h a day in the icu . the control group ( cg ) received conventional therapy for septic shock , namely : infective sources control , early appropriate antibiotics , fluid challenge and vasopressor infusion , and lung protected ventilatory strategy based on surviving sepsis campaign guidelines . continuous renal replacement therapy ( crrt ) in the form of citrate - based postdilution continuous venovenous hemofiltration using polysulfone high flux hemofilter ( f 80 , fresenius medical care , germany ) was provided in the presence of acute kidney injury categorized as injury or more based on risk , injury , failure , loss , and end - stage ( rifle ) criteria . treatment group ( tg ) ( endotoxin hemoadsorption group ) received endotoxin hemoadsorption in addition to conventional therapy . a double lumen 12-f hemodialysis catheter ( arrowguard blue plus antimicrobial catheter , arrow international inc . , usa ) was inserted into either the internal jugular or femoral vein for vascular access by the attending intensivists / physicians immediately after randomization . endotoxin hemoadsorption was performed with alteco endotoxin hemoadsorber using ak10 machine ( gambro - hospal , stockholm , sweden ) at a blood flow rate of 120 - 150 ml / h . each patient received two 2-hourly sessions of hemoadsorption 24 h apart . low molecular weight heparin ( lmwh ) was used for anticoagulation at the discretion of the treating physician , with tinzaparin 1000 iu iv as the default dosage . crrt was started in - between two sessions of hemoadsorption and afterwards based on the same starting criteria for cg if necessary . disease severity and prognosis were assessed with acute physiology and chronic health evaluation ( apache ) iv score . clinical parameters and laboratory data dosage of vasopressor was expressed as vasopressor score ( vs ) using the formula : ( dopamine dose 1 ) + ( dobutamine dose 1 ) + ( adrenaline dose 100 ) + ( noradrenaline dose 100 ) + ( phenylephrine dose 100 ) , wherein all doses are expressed as g / kg / min . dose - response relationship between vasopressor and blood pressure was expressed as vasopressor dependency index ( vdi ) and was calculated using the formula : vs / map . primary end point was the change of the sofa score from 0 to 72 h of randomization . secondary end points included changes of vs , vdi , pao2/fio2 ratio , icu length of stay ( los ) , hospital los , and 28-day mortality . sample size was estimated based on previous study findings . with the power of 80% , type i error probability of 0.05 , mean sofa score difference of 2 , standard deviation of 1.5 , the estimated sample size was 20 . univariate analysis was performed using fisher 's exact test for categorical data or mann - whitney u - test for continuous data where appropriate . the analysis was performed by the statistical package for social sciences for windows , version 16.0 ( spss , chicago , il , united states ) . this study was terminated early by the monitoring committee as an interim analysis could not identify any significant clinical benefit . from february 2010 to june 2012 , 15 patients were recruited ( seven in the lps hemoadsorption group and eight in the cg ) . all patients except one from cg yielded gram - negative bacteria from saved microbiological culture specimens . among them , klebsiella species were the most commonly isolated organisms ( total 40% , tg vs. cg = 29% vs. 50% ) , followed by escherichia coli ( total 33% , tg vs. cg = 29% vs. 38% ) . multiple bacteria were isolated from 27% of cases ( tg vs. cg = 29% vs. 25% ) . adequate , appropriate initial antibiotic coverage ( based on subsequent microbial sensitivity pattern ) were given to 93% of patients within 24 h of recruitment ( tg vs. cg = 86% vs. 100% ) . sofa score showed more obvious improvement among cg group at 48 h and 72 h , but this was not statistically significant [ table 2 ] . both groups showed decreased use of vasopressor over time , but the improvement did not differ between groups . improvement of oxygenation was more obvious among the tg group but did not reach statistical significance . continuous veno - venous hemofiltration was given in all tg patients and 63% of cg patients . for those icu survivors ( six from treatment and control arm respectively ) , no patient required dialysis support within 1 and 3 months after recruitment . concerning the adverse events during alteco endotoxin hemoadsorption , severe thrombocytopenia ( platelet count < 20 10 /mm ) occurred in one patient but no bleeding event was reported . transient hypotension ( map 60 mmhg ) occurred in one patient during the initiation of the first endotoxin hemoadsorption , who required increased vasopressor support . to our best knowledge , the current study is the first rct to investigate the therapeutic effect of this new endotoxin hemoadsorption device ( alteco endotoxin hemoadsorber , alteco medical ab , lund , sweden ) in gram - negative septic shock patients . unfortunately , the study was terminated early by the monitoring committee as an interim analysis showed a low probability of significant findings . extracorporeal blood purification as a treatment for sepsis consists of multiple treatment modalities ; these either targeted inflammatory mediators or bacterial toxins like endotoxins or both . crrt is commonly performed in icu settings for patient with septic acute kidney injury . however , the use of low or normal volume continuous venovenous hemodialysis or hemofiltration failed to demonstrate an improvement of patient outcomes in severe sepsis . high volume hemofiltration ( hvhf ) or pulse hvhf removed cytokines effectively , and initial study showed promising results . however , recently published ivoire study could not identify any significant mortality nor organ function benefit when compared with standard volume hemofiltration . moreover , hvhf incurred an increase of nursing workload ( especially without the use of online treatment modality ) , higher treatment cost due to the use of large volumes of replacement fluid and potential electrolytes / drug concentration disturbances . hemodialysis or hemodiafiltration using high cutoff membrane offers a good cytokine clearance , but significant albumin loss , together with albumin - bound drugs are the key problem which require particular attention . coupled plasma filtration adsorption is a relatively investigational tool , although initial experiences were impressive . hemoperfusion with cytokines and/or endotoxin hemoadsorption columns require simple set up and equipment , which is more feasible in an icu setting . nowadays , there are three different methods for endotoxin hemoadsorption in septic shock which have more clinical experience . pmx immobilized fiber column hemoperfusion ( toraymyxin , toray industries , tokyo , japan ) is the most commonly used device . this device has been used for the treatment of septic shock since 1994 in japan and since 2002 in europe . it has gained popularity worldwide in recent years , especially after the euphas ( early use of pmx b hemoperfusion in abdominal sepsis ) study . a recent meta - analysis by mitaka clearly showed that pmx hemoperfusion treatment had significant beneficial effects on patient hemodynamics , pulmonary oxygenation , and mortality . endotoxins may also be bound to an adsorber contained albumin ( matisse , fresenius medical care , bad homburg , germany ) . trends in the improvement of morbidity and organ dysfunction were found in initial nonrandomized studies . however , a subsequent multicenter rct could not identify any significant clinical benefit , which then limited its clinical use . endotoxin capture by specially designed synthetic peptides is another method ( alteco endotoxin hemoadsorber , alteco medical ab , lund , sweden ) . it is a class iia medical extracorporeal device consisting of a rigid porous matrix which can significantly increase its blood contact area . tailor - made synthetic peptides with a high affinity for endotoxins are connected to the surface of the polyethylene plates with a covalent bonding technique . the clinical experience for this device is scarce and is limited to case reports and case series . showed that the duration of noradrenaline infusion was significantly shorter in adsorber - treated patients compared to controls ( p = 0.03 ) . in our study , vasopressor use decreased nicely in adsorber - treated patients but this also occurred in control patients . compared with the study by ala - kokko et al . , our study cases were older ( 75 vs. 60 years old ) , had more significant organ failure as expressed by sofa score ( 13 vs. 9 ) , were on huge doses of vasopressor ( vs 50.5 vs. 11.1 ) and had much higher predicted mortality ( 65% vs. 27% ) . these findings also apply when compared with pmx hemoperfusion - treated patients in the euphas study . which indicated that our adsorber - treated cases were much sicker . by closely examining the difference between the adsorber - treated patients and the controls , we noted that the control cases had faster organ recovery as expressed by a more rapid drop in sofa score , less vasopressor use , better oxygenation improvement , and lower icu length of stay . although there was no statistically significant difference due to the small sample size , the apache iv score predicted mortality rate was much higher in adsorber - treated patients compared with controls , which may provide a good explanation on the discrepancy in clinical outcome parameters . it is possible that with such severe cases , the addition of endotoxin hemoadsorption offered no further clinical benefit when compared with standard intensive care , although suboptimal organ support or ineffective endotoxin removal could be other reasons for this . concerning the first alternative ( suboptimal organ support ) , the standardized mortality ratio by apache iv risk of death for the adsorber - treated patients was 0.7 which was fair ; this indicated that suboptimal care was less likely . for the second reason , due to great difficulty in sourcing a quantitative endotoxin assay and limited funding , no endotoxin assay was performed . therefore , we could not be sure that the patients had adequate endotoxin removal during hemoadsorption . concerning the side effects of alteco endotoxin hemoadsorption , ala - kokko et al . in fact , thrombocytopenia ( platelet count < 150 10 /mm ) occurred in all adsorber - treated patients but only one case suffered from severe thrombocytopenia ( platelet count < 20 10 /mm ) . no bleeding event was noted , and no platelet transfusion was given . in the case this may be related to the fact that we used lmwh for anticoagulation instead of unfractionated heparin ( ufh ) used in ala - kokko et al . the pharmacokinetics of lmwh are more predictable than ufh , which may be more obvious in septic patients . transient hypotension occurred once and required an increase in vasopressor support , but no arrhythmia was documented , as in the cases reported by ala - kokko et al . the sample size was estimated based on previous studies , but early termination of this study further limited its power . given that there are early reports on the effectiveness of hemoadsorption technique on treatment of septic shock , this negative study could offer readers information on patient 's clinical response and side effect profile of this novel device . for this single - center study , case recruitment proved to be quite difficult as the endotoxin hemoadsorption technique was a new treatment option in our locality . recruitment rate was slow and multiple center collaboration could be the only means to resolve this issue . an endotoxin activity assay ( eaa ) was not performed in our study , similar to the landmark euphas study , due to the unavailability of the point - of - care testing device in our locality . although endotoxin activity reflects the severity of illness in critically ill septic shock patients , its prognostic value is poor . we believed that the availability of eaa result should be a bonus but not a must for this study . however , in order to minimize potential error on cases recruitment , we targeted only those suffering from intra - abdominal sepsis with shock . so far , the microbiological findings have yielded gram - negative bacteria in almost all of the recruited cases . finally , blinding was not possible for this study and may have contributed to further bias . we could not identify any clinical benefit on the addition of alteco endotoxin hemoadsorption to conventional therapy in patients who suffered from intra - abdominal sepsis with shock . the side effect profile of this device was acceptable . given that there are early reports on the effectiveness of hemoadsorption technique on treatment of septic shock , larger multicenter study is indicated to further investigate the potential benefit or drawback of this novel device . a recent report published by the american association of colleges of nursing ( aacn ) noted that over 67,000 qualified applicants were not accepted into baccalaureate and graduate nursing programs in the usa in 2010 . the report also noted that almost two - thirds of the nursing schools participating in the survey noted that faculty shortage is the primary reason for not accepting all qualified applicants into baccalaureate programs . the consequence of a nursing shortage is nurses work longer hours under stressful conditions , which leads to nurses being more prone to making mistakes and medical errors . schools of nursing are increasingly using hospital - based nurses to precept students during clinical rotations . these nurse preceptors extend the faculty at a time when a shortage of nursing faculty limits nursing school enrollment . combined with initiatives already in place , such as using master 's prepared nurses at the hospital as loaned faculty , compressing students ' clinical rotations and assigning clinical rotations to off - shifts or , in less popular nursing units , using nurse preceptors as clinical faculty helps in two ways : it increases the number of available clinical nursing slots and it provides qualified clinical instructors . as little quantitative research on the effectiveness of using preceptors as clinical instructors early in a nursing program has been reported , this study looks at the question given baccalaureate students in their second medical - surgical class , do precepted students perform as academically well as traditionally prepared students ? clinical groups in texas traditionally have a ratio of one master 's prepared instructor to 10 students . the instructor 's role is to monitor students in the clinical setting and instruct them in meeting their educational learning objectives . when class size is extended to increase enrollment , procuring a sufficient number of qualified clinical instructors is often difficult . that nurses qualified for teaching can make higher salaries working as a nurse in a hospital than as faculty in a university exacerbates the faculty shortage problem additionally , as the number of clinical slots dedicated to nursing students is limited , schools in the region compete with each other . students often demand accessible and timely information , and they want flexibility to meet their needs of working , studying , and raising a family . the younger students rely heavily on technology for learning , entertainment , and life scheduling . students precepted one - on - one with registered nurses ( rn ) in hospital settings are more likely to find their needs met than is possible under a traditional group model , which permits less interaction between faculty and student , limits student opportunities for learning and skills practice , and provides an inaccurate view of the profession [ 4 , 5 ] . according to the texas board of nurse examiners , master 's prepared faculty can oversee the teaching activities of 12 rn preceptors , each of whom can supervise two undergraduate students . using preceptors as clinical faculty alternatives more than doubles the number of students ( n = 12 2 = 24 ) than can be placed in traditional clinical rotations ( n = 10 ) . the policy established by the texas board of nursing states that a precepted student must be visited by a faculty member at least once a month . faculty conducting the study rounded on precepted students at least twice a month since the students were early in their nursing program . hospital - based clinical preceptors , as alternatives to clinical faculty , expect adequate support to function within the educator role . according to yonge and myrick , preparation of preceptors includes teaching educational principles that help prepare the preceptors beyond their usual staff nurse orientation . wilkes et al . identified that continued support materials , beyond orientation , were essential to success . using hand - held computers allows preceptors to obtain support at the bedside when time is at a premium and desktop services are not available . the use of hand - held computers with internet access is an effective way for preceptors to obtain information , such as faculty contact information , school policies , and student clinical schedules , prepare anecdotal notes , and search for useful clinical information [ 12 , 13 ] . use of the precepting alternative also frees up additional nurses and hospital units for clinical training ; nurses on smaller units not able to support a traditional group of 10 students could precept one - on - one and nurses working off - shifts can serve as preceptors . students participating in precepted groups find that they have more scheduling flexibility , greater opportunities for learning and practicing skills , and more relevant learning experiences . to prepare the nurses for the preceptor role , the researchers developed a 14-module online preceptor education course . the module units , listed in table 1 , were designed to provide direction on teaching students as adult learners and to promote critical thinking in nursing students . the project team , including faculty and hospital educators , attended the preceptors ' presentations to offer technical training and support . upon completion of the preceptor training , nurses received 9.6 continuing education units . having blackboard access also gave the rn preceptors access to the students ' course materials , including syllabi and lecture notes , which permitted the rn preceptors to provide clinical experiences that met the learning objectives and to keep pace with the students learning progression . as nurses traditionally work with new hire graduates and not students in their second semester of nursing education , it was important that preceptors knew that these students would not perform at the same level as a new graduate nurse . technology assisted in permitting continuous availability of faculty and in forming a communication net for students , preceptors , and faculty . communication was via a dedicated web page , e - mail , cellular phones , traditional pagers , and handheld computers . preceptors working at the bedside could communicate with nursing faculty in the office . using the handheld computers , preceptors had a rapid means of access to relevant nursing and drug information while they were working with the students . in addition , the research faculty and project staff developed a project - specific website to provide quick access to contact information , school policies , performance issue information , tips and topics , and links for emergencies and needle sticks . the website was a resource for preceptors and students whether the students were precepted during school hours or on off - shifts . both preceptors and students could rapidly locate faculty contact information or receive technical support 24 hours a day . this was necessary as the precepted students worked the schedule their preceptor worked , whether this was a day , night , or weekend shift . e - mail group links permitted communication to the project staff and the control and experimental student groups . the availability of student performance information and school policies assisted preceptors to take appropriate action in addressing student attendance , dress and appearance , safety , and professionalism issues . using blackboard to deliver the education course online permitted the rn preceptors to complete the training at their convenience since the students worked all shifts , discussion boards were set up to support communication and submission of assignments . nursing faculty moderated the discussion postings and responded accordingly . additionally , faculty and hospital educators periodically made rounds when students were scheduled to be with their preceptors to stay in contact , answer questions , and provide support . the use of technology helped to decrease preceptor resistance in precepting students without the instructor being present and increased faculty assurance in the quality and relevance of the preceptors teaching . the students had to complete a total of 96 hours in the clinical setting and were permitted to work between 8 and 24 hours per week . for faculty to keep track of student schedule and hours , students posted their clinical schedule at least 48 hours prior to working with their preceptor . faculty held weekly post - clinical conferences with students at the school to answer questions and reinforce learning objectives . however , faculty were available to the students and preceptors by cellular phone twenty - four hours a day . the study used a quasi - experimental design where students ( n = 69 ) were randomly assigned to a control group ( traditional clinical group ) or experimental group ( precepted group ) . the subjects were in the second semester of their nursing education , and instruction that semester included pharmacology , gerontology , pathophysiology , and the second medical - surgical nursing course . both experimental and control students volunteering to participate in the study submitted an informed consent form in accordance with the university 's institutional review board policy . for experimental and control groups , the student 's accumulative numerical course grade in medical - surgical course 1 ( taken in the first semester ) was used as an independent variable . the dependent variables were students ' numerical grades on unit examinations given throughout the semester , comprehensive final examination grades , and accumulative numerical course grades in both the medical - surgical course 2 and the corequisite pharmacology course . both experimental and control students took a standardized medical - surgical exam . although scores earned on exams administered to measure what the student learned in the classroom does not have established reliability and validity for measuring clinical performance , issues surrounding clinical evaluation exist . some of the identified issues pertinent to this study include the subjectivity of the evaluation especially when using novice clinical faculty . novice faculty ( bedside nurses serving as preceptors ) may have limited formal education and experience in evaluation of students and often lack confidence in their ability to fairly evaluate students [ 16 , 17 ] . using a grading rubric , two faculty members on the research team reviewed the precepted student 's nursing process papers at weeks 4 , 8 , and 12 and recorded these grades . to increase interrater reliability , the two researchers critiqued one student 's paper prior to week 4 to standardize scoring . a clinical evaluation form was developed for preceptor use ( table 1 in supplementary material available online at doi:10.1155/2012/276506 ) . after each 12-hour shift , the preceptor evaluated the student 's clinical performance and faxed the form to the research team . if a student is consistently scoring 2 or less on the evaluation , the faculty meets with the preceptor to discuss the weaknesses of the student . faculty would meet with the student as well to discuss strategies for improving their clinical performance . the final clinical evaluation was completed by the faculty and was based on data from the preceptor evaluations , the nursing process paper grades , and the faculty 's impressions when rounding on the student in the hospital . the students were randomized into two groups , experimental or precepted group ( n = 37 ) and control or traditional group ( n = 32 ) . the precepted students were assigned clinical rotations at two tertiary - level hospitals , where they worked one - on - one with a baccalaureate - prepared registered nurse ( rn ) on a medical or surgical unit or a surgical intermediate care unit . as per the request of the two hospitals , precepted students could not work on the days other school of nursing held their traditional clinical . this meant that there was one day a week in which the precepted students could not schedule a clinical day . the control students were mixed within the traditional training groups of 10 students , and these groups were assigned to medical - surgical floors at various local hospitals . the criteria for eligibility were that the rns ( 1 ) have at least one year experience as a registered nurse ; ( 2 ) have a recent satisfactory annual evaluation by their nurse manager ; ( 3 ) have a current bcls and nursing license ; ( 4 ) completed the online preceptor education course ; ( 5 ) graduated from a baccalaureate nursing program . inclusion criteria for students were that they were in their second semester of nursing school and had not previously withdrawn from or failed a class . the students ' final numerical course grade from the first semester medical - surgical course was analyzed using an independent sample t - test to determine whether the first semester grade needed to be used as a co - variant . however , this test showed no significant difference between the experimental and control designated groups . student 2nd semester medical - surgical examination scores ( 4 units and 1 comprehensive final ) were analyzed using a mixed model approach for repeated measures anova . final numerical grades from their pharmacology course and a standardized specialty medical - surgical examination ( hesi ) were analyzed using independent sample t - tests . no significant differences between the experimental and control groups on any measurement were found . for discussion purposes , an implication is that precepted students did as academically well as students in the control group . likewise , this could be stated , as precepted students do no better than traditional students academically despite the one - on - one clinical treatment . given this , the study supports using hospital - based rn nurses as clinical preceptors . using rns as preceptors not only provides much needed clinical faculty but also frees up clinical slots that previously have not been available . the four unit examination grades and the final examination grade were analyzed using a mix model approach for repeated measures anova . there was no statistically significant difference between the precepted and control groups ( f = .936 , df = 63 , p = .449 ) . an independent sample t test was computed to determine if there was a significant difference between precepted and control groups in academic performance as demonstrated by the final course grade students received in their second semester medical - surgical course . students in the traditional clinical group had a mean of 83.7 ( sd = 5.9 ) , and the precepted students had a mean of 83.5 ( sd = 4.6 ) . there was no statistical difference between the two groups ( t = .118 , df = 67 , p = .906 ) . ( hesi , now owned by elsevier ) medical - surgical specialty exam at the end of the semester , this mean score was considered in the analysis . the mean standard score for the traditional students was 784.19 ( sd = 182.5 ) as compared to a mean standard score of 807.76 ( sd = 136.7 ) for the precepted students . however , there was no statistical difference between the two groups ( t = .612 , df = 67 , p = .543 ) . students in the traditional clinical groups usually prepared medication cards the day before each clinical session . as students in the precepted group did not know which patients their preceptor would have until report , they had to take a different approach . they reviewed each medication and presented the information to their preceptor before administering the medication . in analyzing pharmacology final course grades , there was no statistical difference between the two groups ( t = .786 , df = 67 , p = 434 ) . the students in the control group had a mean grade of 89.1 ( sd = 3.8 ) , and the precepted group had a mean of 88.3 ( sd = 4.8 ) . on average , the students in the precepted group received ratings of 35 for the clinical competencies listed on the clinical evaluation form . confident preceptors not only rated the clinical performance , but also provided anecdotal information to justify the given ratings . preceptors did note that the clinical evaluation process was an added responsibility to their busy clinical day . unlike students in the traditional group , precepted students arrived for their shift with no preparatory work in place . the student and their preceptor selected one patient as the student 's primary patient for that shift . although the precepted student was responsible for obtaining the same information as that of a traditional student , the precepted students did this on shift and completed the nursing process papers retrospectively . the faculty found that the quality of the nursing process papers were similar between the precepted and the traditional groups . students in both groups struggled with parts of the nursing diagnosis ( related to statement ) and setting measurable expected outcomes . the precepted students , instructed to turn in their paperwork within a week of completing the shift , tended to fall behind , allowing the paperwork to accumulate . the information technology ( it ) member of the research team worked closely with the two hospitals that participated in the study . the it personnel from the two hospitals and the university had to breech the firewall that existed between institutions . once the two systems were connected , the it member of the research team met with preceptors one - on - one to help them navigate the blackboard site within the university . the it member held mini tutorials to help the preceptors learn how to use their institution 's intranet , access the university 's blackboard site , and navigate the preceptor course . this challenge was not anticipated by the research team and did require additional time not planned in the original proposal . most preceptors had served in the role in the past , orienting new hires on their respective units . one occasion arose where a preceptor placed the student in a dangerous situation ( administering medications without being present ) . the student reported the incident , and working with the institution , the preceptor was replaced with another nurse . although many of the preceptors have mentored new nurses on their units , they had not participated in a formal evaluation process . some preceptors would circle all the same number on the likert scale clinical performance evaluation and would not provide any commentary . the members of the research team would meet with preceptors and ask for a verbal evaluation of the student 's performance . questions asked included an assessment of the student 's ability to perform a focused review of systems and physical exam , knowledge of medications and their administration , and the potential to use the nursing process to plan appropriate care for the patient , for example . reviewing the nursing process papers also provided insights as to how the student was performing in the clinical setting . in the last decade , we have seen an increase in the number of applicants to nursing programs , a decline in the number of nurses in the workforce , and a decline in the number of nurses who pursue a career in academia . with the nursing shortage , demands are made on schools to increase enrollment ; however , with the shortage of nursing faculty , this demand has been difficult to meet . as a barrier to increasing enrollment has been clinical availability , a solution is using nurse preceptors to extend faculty in the clinical setting . this project examined the use of preceptors , supported by training and technology , to facilitate the clinical experience of students in their second semester medical - surgical course . precepting students are not a new concept within nursing ; however , a preceptor support model for students early in their nursing education has not been fully studied . the purpose of the study was to determine whether students who were precepted performed as well as those in a traditional clinical group . as there was no significant difference in performance on grades and hesi scores , the results suggest that from an academic perspective , providing clinical education when using qualified and trained preceptors did not interfere with the student 's ability to master the course content . the quality of the nursing process papers produced by students was deemed to be equal between the two groups . students in both groups were provided feedback on their papers and were asked in equal proportion to resubmit work . as the main problem encountered was the timeliness of the submission of the paperwork , a solution would be to design a mechanism within the computer - scheduling program that locked out students from posting their schedule until all nursing process paperwork was completed .

background and aims : severe sepsis and septic shock are common causes of mortality and morbidity in an intensive care unit setting . endotoxin , derived from the outer membranes of gram - negative bacteria , is considered a major factor in the pathogenesis of sepsis . this study investigated the effect of alteco endotoxin hemoadsorption device on gram - negative septic shock patients.materials and methods : an open , controlled , prospective , randomized , single - center trial was conducted between february 2010 and june 2012 . patients with septic shock due to intra - abdominal sepsis were randomized to either conventional therapy ( n = 8) or conventional therapy plus two 2-hourly sessions of alteco endotoxin hemoadsorption ( n = 7 ) . primary endpoint was the sequential organ failure assessment ( sofa ) score changes from 0 to 72 h. secondary end points included vasopressor requirement , pao2/fio2 ratio ( pfr ) , length of stay ( los ) , and 28-day mortality.results:this study was terminated early as interim analysis showed a low probability of significant findings . no significant difference was noted between the two groups with respect to change in sofa score , vasopressor score , pfr , los , and 28-day mortality . side - effect was minimal.conclusions:we could not identify any clinical benefit on the addition of alteco endotoxin hemoadsorption to conventional therapy in patients who suffered from intra - abdominal sepsis with shock . the side effect profile of this novel device was acceptable . the shortage of nursing faculty has contributed greatly to the nursing workforce shortage , with many schools turning away qualified applicants because there are not enough faculty to teach . despite the faculty shortage , schools are required to admit more students to alleviate the nursing shortage . clinical groups in which preceptors are responsible for student learning extend faculty resources . purpose . to determine the effectiveness of an alternative clinical experience ( preceptorship ) . methods . quasi - experimental , randomized , longitudinal design . students were randomized to either the traditional or precepted clinical group . the clinical experience was a total of 12 weeks . groups were compared according to several variables including second semester exam scores , hesi scores , and quality and timeliness of clinical paperwork . sample . over a two - year period , seventy - one undergraduate nursing students in the second semester medical - surgical nursing course participated . 36 were randomized to the experimental group . the preceptors were baccalaureate - prepared nurses who have been practicing for at least one year . setting . two hospitals located in the texas medical center . statistical analysis . descriptive statistics and independent t - test . results . there was no difference between the groups on the variables of interest . conclusion . students in the precepted clinical group perform as well as those in a traditional clinical group .

ameloblastoma is seen to have a benign appearance on histology , irrespective of its variable clinical behavior . microscopically various patterns have been described ( follicular , plexiform , acanthomatous , papilliferous - keratotic , granular cell type , desmoplastic , vascular , and dentinoameloblastoma).[13 ] majority of patients present in the fourth decade . men are affected slightly more often than female , with a particularly elevated incidence in eastern africa . more than 80% of ameloblastomas arise in the mandible ( mostly angle or ramus ) . they are either primary or secondary soft tissue tumors , the latter appearing after operations . radiologically , ameloblastomas present as unilocular or multilocular translucencies . malignancy in the ameloblastoma has been divided into two distant lesions . a malignant ( metastasizing ) ameloblastoma is diagnosed when a seemingly histologically benign ameloblastoma produces a metastasis resembling the original lesion . ameloblastic carcinoma is an odontogenic tumor having the overall microscopic architectural features of ameloblastoma but in addition having malignant cytological features such as marked nuclear atypia and numerous mitotic figures . in the year 1965 , tsukada et al . two years later , a case of granular cell ameloblastoma with metastasis to cervical vertebrae was reported . we reviewed the literature for any cases of granular cell ameloblastoma with metastasis , which might have been reported from year 1967 until now . to the best of our knowledge a 40-year - old female first reported to the surgery department of safdarjung hospital , new delhi , india . she presented with a disfiguring swelling on the right side of neck of approximately two years duration [ figure 1 ] . roentgenogram of the mandible showed swollen translucent cystic structures reaching from corpus to ramus of the right mandible . excision of zygomatic arch and surrounding soft tissue , masseter muscle , and temporalis muscle along with right parotid , was done . reconstruction was performed using right pectoralis major myofacial flap for buccal mucosal reconstruction and delto pectoral flap with split skin graft for skin defect . two years following surgery , patient 's follow up with x - rays , have not revealed any new lesions . patient with a large abnormal swelling on the right side of face gross examination of the excised specimen showed a well circumscribed large pink tan growth measuring 7.5 5 4 cm in the region of the mandible . tumor cells formed nests supported by fibrous connective tissue stroma [ figure 2 ] . at the periphery of the nests , the inner cell mass showed round to polygonal cells with abundant granular cytoplasm and small pyknotic nuclei [ figure 4 ] . one lymph node showed tumor metastasis characterized by the presence of granular cells [ figure 5 ] . ( h and e , 200 ) cells displaying cytoplasmic granularity with pyknotic nuclei , some of which are eccentric . ( h and e , 400 ) metastasis in lymph node showing tumor cells with granular cytoplasm . gross examination of the excised specimen showed a well circumscribed large pink tan growth measuring 7.5 5 4 cm in the region of the mandible . tumor cells formed nests supported by fibrous connective tissue stroma [ figure 2 ] . at the periphery of the nests , the inner cell mass showed round to polygonal cells with abundant granular cytoplasm and small pyknotic nuclei [ figure 4 ] . one lymph node showed tumor metastasis characterized by the presence of granular cells [ figure 5 ] . ( h and e , 200 ) cells displaying cytoplasmic granularity with pyknotic nuclei , some of which are eccentric . ( h and e , 400 ) metastasis in lymph node showing tumor cells with granular cytoplasm . the tumor is made up of proliferating odontogenic epithelium especially of enamel organ - type tissue that has not undergone differentiation to the point of hard tissue formation . it has been postulated that the epithelium of origin is derived from one of the following sources : ( 1 ) epithelial lining of odontogenic cyst , ( 2 ) dental lamina or enamel organ , ( 3 ) disturbances of developing enamel organ , ( 4 ) basal cells of surface epithelium , or ( 5 ) heterotopic epithelium of other parts of the body . our patient presented with a metastasis of granular cell ameloblastoma at the first instance , giving a brief history of 2 years . most common sites of metastasis are lung ( 76.7% ) , followed by regional lymphnodes ( 37.8% ) , pleura ( 16.2% ) , vertebrae ( 13.5% ) , skull ( 10.8% ) , diaphragm ( 8.1% ) , liver and parotid ( 5.4% ) and even more rarely , the spleen and kidney . granular cells of granular cell ameloblastoma are clearly of epithelial origin staining exclusively for cytokeratin . the term granular cell ameloblastic fibroma is a misnomer , as a number of these cases are probably central odontogenic fibromas exhibiting granular cell change . ultrastructurally it has been revealed that it is the lysosomal overload in these cells that imparts the characteristic granularity . whether granular cell change in ameloblastoma is a degenerative process or a harbinger of a more aggressive course is a matter of debate . all three cases of granular cell ameloblastoma ( our case included ) have eventually produced metastasis . add to this , our patient gave a short history of duration before tumor metastasis . although the exact implication of a granular cell change in ameloblastoma can not be ascertained due to paucity of cases documented , yet reported cases must be followed closely in anticipation of metastasis . mesenteric and omental mesothelial cysts are responsible for only 1 in 100,000 of adult hospital admissions , and are thus left low on the list of differential diagnoses for abdominal pain . further evaluation of such masses is first directed at determining a tissue origin if possible . lymphangioma accounts for the most commonly reported of such lesions , followed by enteric cyst , enteric duplication cyst , non - pancreatic pseudocyst , and mesothelial cyst . gastrointestinal leiomyomas ( cystic spindle cell tumors ) in particular have been reported to undergo central liquefactive necrosis and hemorrhage , appearing as cystic mesenteric or omental lesions on imaging . the pathogenesis of a mesothelial cyst involves failed coalescence of mesothelial - lined surfaces , typically involving the small bowel , mesentery , or mesocolon . imaging usually demonstrates a fluid - filled cavity without a readily identifiable wall . unlike lymphangiomas , however , given the overlapping features of such cystic intra - abdominal masses on imaging , definitive diagnosis is made on thorough histopathologic analysis of the resected specimen . a 41 year - old male presented to our emergency department with periumbilical abdominal pain and associated intermittent nausea and vomiting of several days duration . medical history was significant only for hypertension , and he was noted to have had prior appendectomy and posterior spinal fusion . physical examination revealed an obese abdomen with localized tenderness to palpation over the mid - abdomen without peritoneal signs . initial emergency department laboratory workup included cbc and cmp , which revealed a hypokalemic hypochloremic metabolic alkalosis . this patient was admitted to the general surgery service and treated conservatively with iv rehydration and empiric antibiotics . accounting for his chronic diarrhea , chief differential diagnosis at that time included sclerosing mesenteritis , small bowel diverticulitis , and small bowel carcinoid tumor . 5-hiaa , octreotide scan , and chromogranin a were within normal limits , lowering suspicion for neuroendocrine tumor . with conservative management , he initially reported improvement in his abdominal pain ; however , his pain never completely resolved and he eventually reported postprandial exacerbation . on hospital day 13 , the decision was made to perform diagnostic laparoscopy with potential mass and/or small bowel resection . the mass present on ct was identified intraoperatively in the mid - jejunal mesentery after an adhesion between two adjacent loops of jejunum was separated ( fig . the mass was resected along with a 15 cm segment of the involved jejunum , followed by primary anastomosis . his postoperative course was uneventful , and he was discharged shortly thereafter with resolution of his pain . on clinic follow - up , he has remained without pain . h&e staining of the specimen demonstrated spindle cells and signs of coagulation necrosis with ghost cells showing prominent eosinophilic staining fig . cd117 and dog1 staining were negative ( not shown ) , excluding gastrointestinal stromal tumor . h&e staining of the specimen demonstrated spindle cells and signs of coagulation necrosis with ghost cells showing prominent eosinophilic staining fig . cd117 and dog1 staining were negative ( not shown ) , excluding gastrointestinal stromal tumor . mesenteric and omental cystic masses are uncommon , representing only 1 in 100,000 acute adult hospital admissions , although it is possible they occur with greater frequency but are missed or identified incidentally as most remain asymptomatic . further hindering their rapid diagnosis when symptomatic is their very nonspecific presentation including but not limited to abdominal pain which is often chronic in nature , mass , or distension . this patient presented with the additional complaint of nausea and vomiting , and it is likely that local mass effect was involved , as concluded by other authors reporting on similar intra - abdominal cystic masses . the exact etiology underlying the necrosis of this patient s mesothelial cyst and its contribution to his abdominal pain is unclear . it is likely that this patient s cyst simply outgrew its own perfusion , although a possible contribution by some physiologic stressor is worth consideration . the mainstay of treatment for symptomatic lesions is complete surgical excision , , as recurrence of incompletely excised cysts has been noted by other authors . fortunately for this patient , surgery seems to have been curative as he remains well on follow - up . rare etiologies of gastrointestinal upset and abdominal pain , while reasonably left low on the differential list , ought not to be excluded from it completely . unfortunately however , it appears that mesothelial cyst and other intraabdominal cystic masses often fail to reveal themselves on physical examination and leave no characteristic clues in recent patient history , only becoming apparent nonspecifically on advanced imaging . the routine practice of ct imaging and the low threshold employed at some institutions for its use is a matter of controversy outside the scope of this case report ; however in the case of our patient , it was diagnostically important , appropriately directed treatment towards surgical management , and led to a therapeutic outcome . routine histopathological examination of resected tissue specimens is also of value in excluding malignancy , arriving at a specific diagnosis and confirming total resection of lesions that are likely to recur if incompletely excised . walter beversdorf , ms-4 : writing of case report , editing , proofing , submitting . this research did not receive any specific grant from funding agencies in the public , commercial , or not - for - profit sectors . written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request .

ameloblastoma is a slow growing odontogenic epithelial tumor of jaw . it accounts for 1% of all tumors and cysts arising in maxilla and mandible . although it is locally invasive and has a marked tendency to recur , metastasis is rare . of the various histological patterns of ameloblastoma , the granular cell type is extremely rare accounting for 4% of ameloblastomas . we report a case of granular cell ameloblastoma with metastasis to the cervical lymph node presenting in a 40-year - old indian female . highlightsmesenteric cystic masses account for a very small fraction of abdominal pain cases.symptoms arise likely as a result of mass effect , even when these lesions are small.on detection of these lesions by imaging , it is important to exclude neuroendocrine tumors.complete surgical excision is curative of symptoms caused by local mass effect.histopathological examination of specimens is the definitive means to differentiate between such lesions , that appear similarly on imaging .

in the past , a number of studies1,2 have given insight into the epidemiology of primary open - angle glaucoma . for caucasians , it is estimated that almost 2% of the population will suffer from the disease , and for afro - caribbeans this percentage goes to almost 8% . according to the world health organization , age - related cataract causes 48% of world blindness . in the united states , age - related lenticular changes have been reported in 42% of those between the ages of 52 and 64 years , 60% of those between the ages of 65 and 74 years , and 91% of those between the ages of 75 and 85 years.3,4 it is therefore almost inevitable that a glaucoma patient will sooner or later need to undergo cataract surgery independently from his chronic disease . the objective of the following systematic review is to examine whether there is strong evidence in order to draw safe conclusions on the long - term survival of a trabeculectomy performed before phacoemulsification.57 we looked for matched studies that compared in a prospective or retrospective way the adverse events after a trabeculectomy alone versus phacoemulsification after an uncomplicated previous trabeculectomy . no limits were applied for language , and we were prepared to translate foreign papers as needed . this search was applied to medline , scopus , embase , the web of science ( science citation index ) , and the cochrane database . in addition , we searched the online contents of the british library and abstracts presented at annual meetings of the association for research in vision and ophthalmology ( arvo ) . we used the following search terms to search all trial registers and databases : randomized controlled trial , controlled clinical trial , random allocation , double - blind method , matched study and trabeculectomy failure , glaucoma filtering operation failure , bleb failure and cataract surgery or phacoemulsification . disagreements were resolved by discussion between the two ; if no agreement could be reached , a third author would decide . in designing the study protocol nonetheless , no author was able to identify one , and therefore , the study design had to be changed . information was extracted from each included study on : ( 1 ) characteristics of the study participants and the inclusion and exclusion criteria ; ( 2 ) type of intervention ; ( 3 ) type of outcome measure ( including visual acuity , intraocular pressure [ iop ] , number of antiglaucoma drugs used , or need for a secondary antiglaucoma surgery ) . to ascertain the validity of eligible studies , a pair of authors working independently determined the adequacy of allocation . to explore variability in study results ( heterogeneity ) , we specified the following hypotheses before conducting the analysis . we hypothesized that effect size may differ according to the methodological quality of the study . the primary outcome measure was the statistical significance of the progressive iop modification before and after phacoemulsification in eyes with previous trabeculectomy . two abstracts from the arvo meetings did not provide full texts , which were necessary to assess the eligibility of the studies , and were not included . all of the studies finally selected for the review were nonrandomized matched studies published in english . the secondary and additional outcomes considered were the number of antiglaucoma medications used and the bleb morphology . the timing of outcome measures was variable and could include monthly investigations , evaluations every 3 months , or a single final evaluation . the first clinical study assessed was from wang et al.8 the main outcome measures for this study were the iop variation after the phacoemulsification in trabeculectomized eyes and the bleb morphology . the study included 27 eyes and concluded that cataract surgery adversely affects trabeculectomy , especially in cases where there is already an initial failure of the bleb . inal et al9 published a controlled study that prospectively assessed completed phacoemulsifications in trabeculectomized eyes as compared to isolated cases of trabeculectomy . the study and control group each comprised 30 patients with primary open - angle glaucoma . the study demonstrated that the success of filtering surgery gradually diminished , and the degree of iop control worsened progressively in a time - dependent manner . in 2005 , ehrnrooth et al10 conducted a retrospective study in order to evaluate risk indicators for cataract surgery and the effect of phacoemulsification on iop control in eyes that had undergone trabeculectomy . from a pool of 138 trabeculectomized patients , the conclusion of the study was that generally the iop is maintained stable after cataract surgery , even though there can be cases of late bleb failure . klink et al11 conducted a prospective study in 2005 with 30 cases and 36 controls . this prospective study concluded that cataract extraction using clear cornea phacoemulsification may be associated with partial loss of the previously functioning filter and with impairment of filtering bleb morphology . shingleton et al7 conducted a retrospective analysis of the surgical results in his private practice , evaluating the iop , best - corrected visual acuity ( bcva ) , and glaucoma medication requirements in patients who underwent phacoemulsification with preexisting glaucoma filters . the study included 58 patients , some of whom had had their cataracts removed through a scleral tunnel or temporal clear cornea . the conclusion was that temporal clear corneal or scleral tunnel phacoemulsification in the setting of a preexisting glaucoma filter was associated with improved bcva , a small but statistically significant increase in iop , and stability in the number of glaucoma medicines required for iop control . a prospective matched study was also conducted by casson et al.12 two groups comprising 28 patients each were tested for the same working hypothesis , comparing the mean iop 1 and 2 years after phacoemulsification , and the surgical success rate in each group was determined by kaplan meier survival analysis . the authors concluded that phacoemulsification may jeopardize long - term iop control in individual patients . rebolleda and muoz - negrete13 also contributed by designing and executing a prospective , nonrandomized comparative ( self - controlled ) study aiming to evaluate the effect of phacoemulsification on iop control in eyes with a previous functioning filtering bleb in patients who were not taking glaucoma medications . their conclusions were that phacoemulsification significantly increased the iop and the number of glaucoma medications necessary in eyes with preexisting functioning filtering blebs . eyes with higher iops before phacoemulsification exhibited worsened postoperative iop control and bleb failure . in their retrospective study , mietz et al14 concluded that cataract surgery had no markedly negative effect on iop . donoso and rodriguez15 conducted a retrospective study comparing two groups of patients treated with 5-fluorouracile ( 5-fu ) . the conclusion was that combined cataract and glaucoma surgery with intraoperative 5-fu was associated with good long - term iop control . this study focused on the effect of phacoemulsification on trabeculectomy survival and therefore the progression of glaucoma . this represents a good example of whether phacoemulsification adversely affects trabeculectomy over the long term . a study by caprioli et al16 assessed the safety of temporal clear corneal phacoemulsification in eyes with previous trabeculectomy . the authors concluded that cataract surgery by temporal clear corneal phacoemulsification in eyes with filtering blebs after trabeculectomy does not adversely affect long - term iop control . park et al17 conducted a retrospective case control study where they attempted to assess the effect of temporal clear corneal phacoemulsification on iop in eyes with a prior trabeculectomy . after a careful statistical analysis , the authors concluded that cataract surgery by temporal clear corneal phacoemulsification in eyes with filtering blebs does not adversely affect long - term iop control . another study that we assessed was published by swamynathan et al.18 in this retrospective case - control study , the main outcome measure was the iop before and after phacoemulsification , at various times postoperatively . this study showed that temporal corneal phacoemulsification can affect the long - term iop control after trabeculectomy with 5-fu or mitomycin c. halikiopoulos et al19 published a prospective case - control study where 68 trabeculectomized patients underwent extraction of their cataracts , either by phacoemulsification or by the extracapsular method . the conclusion of this study was that in the long term , the complication rate of the phacoemulsification was not statistically significant in respect to the extracapsular method with regard to the glaucoma progression . a review of the literature resulted in a large number of potentially eligible studies that needed to be assessed for inclusion against predetermined criteria ; only a small proportion of these were eventually included in the review ( tables 1 and 2 ) . the process for selecting the studies was explicit and conducted in such a way as to minimize the risk of errors and bias . the process by which decisions on the selection of studies were made was specified in a preliminary meeting of the authors that clarified these issues , including who would carry out each stage and how it would be performed . the aim of the selection process was to ensure that all relevant studies would be included in the review . as already known , the types of study used to assess the effects of interventions can be arranged into a hierarchy , based broadly on their susceptibility to bias . although the rcts are considered the best study design with which to evaluate the effect of an intervention , in cases where it is unworkable or unethical to randomize participants , a quasi - experimental or an observational design may instead have to be used . simply grading studies using this hierarchy does not provide an adequate assessment of study quality , because it does not take variations in quality among studies of the same design into account . even rcts can be implemented in such a way that findings are likely to be seriously biased and therefore of little value in decision - making . therefore , it is important to consider the individual aspects of the study design that may introduce bias rather than focusing on the descriptive label used . wang et al8 presented a prospective study where preoperative iop and two parameters of the ultrasound biomicroscopy images , including visibility of a route under the scleral flap and reflectivity inside the bleb , were evaluated for an association with postoperative success rate , using kaplan meier survival analysis . meier curve is that the method can take some types of censored data into account , particularly if a patient withdraws from a study , ie , is lost from the sample before the final outcome is observed . the same statistical method was also used by mietz et al.14 in the study by inal et al,9 the data were collected retrospectively , even though success rates were investigated by kaplan meier survival analysis , and the factors influencing final success were submitted to logistic regression analysis . this latter approach is used extensively in the medical sciences to predict a patient s propensity to a certain event . the study by ehrnrooth et al10 was a retrospective case - control investigation ; the paper from klink et al11 and the case - control study by halikiopoulos et al19 were prospective studies . their statistical analysis was based upon standard deviation calculation and extrapolation of the clinical statistical significance ( p - value ) , which limited the strength of the results . shingleton et al7 used the same statistical methods , but also introduced the best way to clinically assess bcva using the logmar scale . the prospective , matched study by casson et al,12 the prospective , nonrandomized comparative ( self - controlled ) study by rebolleda and muoz - negrete,13 and the retrospective case - control study by caprioli et al16 used the same statistical methods , with the limitations mentioned above . a very good impression comes from the study by donoso and rodriguez.15 they used the log - rank test ( sometimes called the mantel cox test ) , which is a hypothesis test to compare the survival distributions of two samples . it is widely used in clinical trials to establish the efficacy of a new treatment compared to a control treatment when the measurement is the time to event ( such as the time from initial treatment to iop spikes ) . lastly , another source of major bias in the finalization of the results is that during the execution of the trabeculectomy , some surgeons used different antimetabolites and some used none , such as in the studies of park et al17 and swamynathan et al.18 it is difficult to assess the influence of antifibrotic therapy on the long - term survival of a trabeculectomy when associated with phacoemulsification because statistical variables increase exponentially and add further to the scientific dilemma . in conclusion , our systematic review sought to gather all the available evidence in favor or against the question of whether phacoemulsification should be performed with or after a trabeculectomy . in the united states , 166 million dental restorations were placed in 2005,1 and clinical studies suggest that more than half were replacements for failed restorations.2 it is anticipated that the emphasis on replacement therapy will increase with the phasing out of dental amalgam . global concerns regarding mercury in the environment are the primary driver for the discontinuation of dental amalgam . identified as one of the top five mercury - added products , dental amalgam is ranked fifth behind batteries , measuring devices , electrical switches and relays , and mercury - containing light bulbs.3 resin composite is the most common alternative to dental amalgam,4 but numerous studies report that composite restorations have more recurrent caries , higher failure rates , and increased frequency of replacement.2,410 simecek et al reviewed the dental records of more than 3000 patients and concluded that there was a significantly higher risk of replacement for posterior composite restorations as compared to amalgam.4 in a study of posterior restorations placed by 243 norwegian dentists , failed amalgam restorations had a mean age of about 11 years , while the mean age of failed composite restorations was statistically significantly lower at 6 years.8 a study of composite and amalgam restorations in the pediatric population indicated that the need for additional treatment was 50% greater in children receiving composite restorations.11 depending on factors , including the size of the restoration , tooth location , and patient type , the lifetime of large to moderate posterior composite restorations is approximately one - half that of dental amalgam.12 the use of composite to restore form and function for posterior teeth damaged by disease , age , or trauma is gaining wide acceptance by the dental community . a myriad of factors can influence the clinical success of class ii composite restorations . clinical parameters , including patient characteristics , tooth preparation , matrix utilization , and composite composition the popularity and demand for resin - based posterior restorations has been increasing steadily since the introduction of these materials in the mid-1950s . the societal focus on aesthetics as well as the worldwide move toward eliminating amalgam restorative materials has contributed to this phenomenon.3 unfortunately the success and/or failure of resin - based composite restorations is dependent upon variables that may be difficult for the operator to control . for example , restorations placed in patients with high caries risk have restoration failure rates two times those of patients with low caries risk.13 these findings have been documented in the adult as well as the pediatric dental patient population.14 clinical data indicate that regardless of which preparation design is adopted or the type of posterior resin - based restoration that is utilized , the practitioner must give careful consideration to the caries status of the patient and adjust recommendations for restorative materials accordingly . posterior resin restorations have been indicated for various types of tooth preparations . in particular , resins are utilized to maximize aesthetics and minimize the loss of tooth structure during preparation . due to the location of the caries and thus the need to restore proximal surfaces in class ii restorations , a number of tooth preparation designs have been advocated . the underlying goal of all of these tooth preparation designs is a reduction in the loss of sound tooth structure . the tunnel technique , as reported by hunt15 and knight,16 has been used to remove proximal caries while leaving the marginal ridge intact . although potentially promising , the lack of long - term clinical studies limits wide adoption of this technique.17 the ability to access and restore a proximal carious lesion directly represents the most conservative proximal restorative technique available.17 this technique is relatively successful in preserving intact tooth structure ( figures 1 and 2 ) . minibox or slot preparations for the restoration of proximal lesions in posterior teeth have also been recommended by clinicians and researchers . these preparation designs have been described as minimally invasive and relatively successful with a reported 70% success rate over an average of 7 years.18 the aforementioned tooth preparation designs successfully limit the removal of sound tooth structure and take advantage of appropriate etching techniques in bonding to intact enamel and dentin . however , depending upon the location and extent of the caries , traditional preparation designs , which involve access through the carious marginal ridge and the removal of infected occlusal enamel and dentin , may be required . these more invasive preparations are indicated in this clinical situation ( figure 3 ) and are well documented in the literature.19 whenever possible , conservative structure - sparing preparation techniques should be used . when restoring proximal surfaces with resin - based composite . considerable attention has been devoted to the relationship between cavity type , cavity size , number of surfaces restored , and the risk of restoration failure . as the number of restored surfaces increases , the risk of restoration failure also increases.2022 for example , as reported in the 2012 review by demarco et al,23 single - surface and class i restorations are less likely to fail as compared to multisurface restorations , and class ii restorations . to minimize restoration failure and mitigate the effects of bonding multiple tooth surfaces , most clinical strategies have focused on methods to decrease the ratio between the bonded surface area to the nonbonded surface area , also described as the cavity configuration or c - factor . the higher the c - factor the less chance for relaxation of polymerization shrinkage . some studies have indicated that the increase in c - factor is also associated with decreased bond strength.24,25 however , recent investigations have suggested that this finding may not be valid for the newer low - shrink resin - based composites.26 along with preparation design and extent of tissue removal , the position of the tooth in the mouth directly influences the overall clinical performance and longevity of the restoration . studies suggest that restorations placed in premolars fail less often than similar restorations placed in molars.20,21 intuitively this finding makes sense in that the masticatory forces and stresses placed on restorations in molar teeth are higher than those placed in premolars . nonetheless , the findings in terms of tooth position and number of restored surfaces indicate that clinicians should utilize posterior resin composites in areas where aesthetics is deemed essential and should maintain as much tooth structure as possible . figures 4 and 5 illustrate the aesthetic results obtained when replacing a proximal amalgam restoration with a resin - based composite restoration . the techniques used to fill and cure resin - based composites , particularly in areas of high masticatory stresses , have received considerable attention . the debate among researchers as well as practitioners regarding bulk cure versus incremental cure continues . incremental filling techniques ( figure 6 ) have long been recommended due to the polymerization shrinkage associated with dental composites . reducing the volume of composite that is polymerized at each stage of the restorative procedure minimizes shrinkage and maximizes the conversion of monomers to polymer . this is achieved , in part , by decreasing the attenuation of the curing light.27 while incremental filling techniques have been taught and utilized for decades , some studies indicate that incremental filling of resin - based composites produces higher shrinkage stress.27,28 in direct contrast , more recent studies report that incremental filling produces lower shrinkage stress when compared to bulk filling techniques.29,30 these diverse and contradictory conclusions are likely due to different testing methods.31 currently , manufacturers are striving to produce resin - based composite systems that have less polymerization shrinkage ( < 2% ) and , more importantly , reduced polymerization shrinkage stress . strategies to improve shrinkage include utilizing new low - shrinking monomers or those with an increased molecular weight.26 as the low - shrinking composite resins improve , incremental filling and curing of posterior composites may no longer be recommended . however , until the long - term clinical success of the lower shrinking composite resin systems is confirmed , using an incremental filling technique in deep cavity preparations is recommended.26 the influence of matrix type on the quality of the proximal contact and the ease of placement of class ii resin restorations has also been evaluated . the ability to reproduce an appropriate , functional , proximal contact with a class ii resin restoration is important to minimize food impaction and thus maintain healthy periodontal tissues . open margin through which oral fluids , eg , saliva , enzymes , water , and cariogenic bacteria , may penetrate . this marginal leakage can lead to recurrent caries , which is the most often cited reason for composite restoration failure.2,410 manufacturers have introduced various types of matrices into the dental market with the goal of affecting or influencing the direction of composite shrinkage during polymerization.32 the literature no longer supports the concept of directional polymerization,33 but these matrices still exist . although there are a myriad of different shapes and sizes , the majority of matrices fall into one of two basic types : ( 1 ) metal matrices , which are straight or circumferential / precontoured and ( 2 ) transparent matrices which are either straight or circumferential / precontoured . despite the theory that transparent matrices will enhance polymerization at the gingival margin , the recent literature suggests that the choice of matrix does not influence the clinical success of class ii posterior resins.32 in addition to matrix type , there are numerous tooth separation ( wedging ) products and techniques . the literature suggests that the type of matrix material / wedge does not influence the clinical performance of class ii composite restorations.34 however , the literature does indicate that no matrix / wedge combination can accurately reproduce an intact proximal surface contact at the precise location of the natural intact tooth.35 researchers and industry continue their efforts to modify composite resin restorative materials in order to improve their handling characteristics , mechanical and physical properties , and clinical performance . the majority of the current resin composites have mechanical properties that make them suitable for use in all areas of the mouth . the functionality of these restorations , however , in areas of high masticatory stress is still a concern . resin restorations that are placed in areas of high function are more prone to exhibit excessive wear and/or marginal fracture despite the advances in the current materials . clinicians must exercise caution when placing large resin - based composite restorations in areas of high function . the longevity of posterior resin restorations placed in patients who have a history of clenching or grinding may be particularly limited.35 while resin composition , tooth preparation design , and matrix systems may influence the lifetime of posterior composite restorations , the primary factor in the clinical failure of moderate to large composite restorations is secondary caries at the margins of the restorations.8 as an example , in a study of radiographs from 459 adults , age 1819 years , the investigators reported that , among interproximal restorations , the failure rate as a result of secondary or recurrent caries was 43% for composite as compared to 8% for amalgam.7 in a separate study of amalgam and composite restorations placed in 812-year - old children , the primary reason for failure of both materials was secondary caries , but secondary caries was 3.5 times higher in composite restorations.5 an increase in secondary caries at the margins of composite restorations suggests that the seal at the composite tooth interface is not adequate to resist the physical , chemical , and mechanical stresses that are present in the mouth . the failure of moderate to large composite restorations has been linked to the degradation of the bond at the tooth surface composite material interface12,3641 and an increase in the concentration of the cariogenic bacterium streptococcus mutans at the perimeter of these materials.4246 degradation of the bond at the interface between the tooth and composite has been associated with the failure of adhesives to form an impervious seal with the dentin.2,4150 failure of the adhesive / dentin ( a / d ) bond leads to open pores at the composite tooth interface and bacterial enzymes , oral fluids , and even bacteria can penetrate these open pores.51 data from in vivo and in vitro studies indicate that the infiltration of these agents into the voids between the tooth and composite will lead to recurrent caries , hypersensitivity , and pulpal inflammation.41,47,52,53 results from clinical studies indicate loss of retention , poor marginal adaptation , and marginal discoloration when the a / d interface is exposed to the oral cavity.54 effective mechanical bonding between the composite restoration and treated enamel has been achieved using appropriate acid - etching protocols , but failure of the bond at the a / d interface threatens the long - term clinical survival of moderate to large posterior composite restorations.39,41,43,52,5557 bonding failures have been commonly tracked to the gingival margin of class ii composite restorations.58 a separation between the composite material and tooth surface has been noted at the gingival margin.55 in class ii composite restorations , there is generally little enamel available for bonding at the gingival margin ; therefore , the bond at this site depends on the integrity of the seal formed with dentin . gaps at the gingival margin have been attributed to unreliable dentin bonding.55,57 in a study comparing the microtensile a / d bond strength of gingival and proximal walls of class ii composite restorations , the adhesive bond to the gingival wall was significantly weaker.59 a complementary spectroscopic study reported a twofold difference in the extent of dentin demineralization at the proximal and gingival margins.50 the difference in demineralization suggests less mineralized dentin at the gingival margin . the cumulative effect of less mineral , increased density , and size of the tubules60 would mean faster and deeper etching at the gingival margin as compared to the proximal wall . although the etch was deeper at the gingival margin , there was considerably less adhesive infiltration of the demineralized dentin matrix at the gingival margin.50 the discrepancy between etching depth and adhesive infiltration led to a large area of exposed collagen at the gingival margin . yoshiyama et al suggested that the increased number of tubules per unit area at the gingival margin would promote efficient adhesive infiltration at this margin.61 however , other variables , including water content , interfere with efficient adhesive infiltration at the gingival margin . water content is higher in dentin at the gingival margin as compared to the proximal wall . water content is increased because of the water present within the demineralized dentin matrix and patent tubules that contain a great deal of dentinal fluid . the presence of this fluid contributes to the contamination of the prepared surface.62 the increased water leads to reduced adhesive infiltration and lower monomer / polymer conversion of the adhesive at the gingival margin as compared to the proximal wall.50 the impact of water upon the effectiveness of bonding is further supported by in vitro investigations that indicate that adhesive monomers or oligomers and unprotected collagen at the gingival margin of class ii composite restorations undergo hydrolytic degradation after 90 days of aqueous storage.49 wet bonding techniques were introduced in the early 1990s to counteract the problems noted with collagen collapse following drying of the demineralized dentin matrix.6366 wet bonding means that the demineralized dentin matrix is fully hydrated throughout the bonding protocol . using this procedure , the channels between the demineralized dentin collagen fibrils are filled with water , solvent , conditioner , and/or oral fluids.67,68 adhesive must diffuse into the fluid - filled spaces of the substrate and along the collagen fibrils . ideally , the solvent in combination with hydrophilic monomers , eg , hydroxyethyl methacrylate ( hema ) conditions the collagen to remain expanded during adhesive infiltration . however , hema , a primary component in many single - bottle , commercial , dentin adhesives , can dramatically reduce the evaporation of water.69 hydrophobic monomers , such as 2,2-bis[4(2-hydroxy-3-methacryloyloxy - propyloxy)-phenyl ] propane ( bisgma ) , would resist diffusing into these sites where there is residual water.7072 in the in vivo situation , there may be little control over the amount of water left on the tooth . thus , it is possible to leave the dentin surface so wet that the adhesive physically separates into hydrophobic- and hydrophilic - rich phases.71,73,74 indeed , results from laboratory investigations indicate that excess moisture prohibited the formation of an impervious structurally integrated a / d bond at the gingival margin of class ii composite restorations.49,50 under clinical conditions , dentists must routinely attempt to bond to naturally wet substrates , eg , caries - affected dentin75 or deep dentin.7679 the water content of caries - affected dentin is 2.7 times greater than that of normal dentin.75 exposed tubules account for 22% of the surface area in deep dentin . in contrast , exposed tubules account for 1% of the surface area of dentin close to the dentino - enamel junction.80 the large increase in exposed tubules in deep dentin means that pulpal fluid will contribute additional moisture to that already present within the demineralized dentin matrix . with the sensitivity of our current adhesives to excess moisture , it is obvious that bonding to these clinically relevant substrates is a formidable challenge.79,8183 this difficulty highlights the potential limitations in utilizing resin - based composites to restore large , deep , carious lesions . water blisters that form in adhesives placed on overly wet surfaces8486 and adhesive phase separation that leads to very limited infiltration of the critical but hydrophobic dimethacry - late component71,87,88 are two examples of the sensitivity of our current adhesives to excess moisture . the optimum amount of wetness varies as a function of the adhesive system.89 it is impossible to simultaneously achieve uniform wetness on all of the walls of the cavity preparation.90 in short , wet bonding is a very technique - sensitive procedure . optimum bonding with our current commercial dentin adhesives occurs over a very narrow range of conditions , eg , water content.78 strategies to promote bonding of the resinous materials to intrinsically wet dentin substrates include the incorporation of ionic and hydrophilic monomers into the adhesive.91 these adhesives etch and prime simultaneously , thus addressing the problems of collagen collapse and simplifying the bonding protocol . the hydrophilicity of these adhesives enhances water sorption , which can lead to hydrolytic breakdown in the mouth.85,90,92 with these systems , the bonded interface lacks a nonsolvated hydrophobic resin coating . the hybrid layers made with these adhesive systems behave as semi - permeable membranes ; water is transferred throughout the bonded interface even after adhesive polymerization.54 the increase in the concentration of hydrophilic monomers in these systems has been associated with decreased structural integrity at the a / d interface.54,93 deterioration of the a / d bond formed with these systems was noted after 1 year of in vivo aging.94 these results suggest that hydrophilicity and hydrolytic stability of resin monomers are generally antagonistic.90 when measured immediately , dentin - composite bonds are generally considered adequate to tolerate conditions in the mouth , but these bonds deteriorate with time . the two major mechanisms of deterioration are fatigue and hydrolysis.95 fatigue has been linked to stresses transmitted to the bond by occlusal forces , thermal expansion and contraction , and polymerization shrinkage of the composite . chronic deterioration of the dentin - composite bond is also related to hydrolysis and leaching of the adhesive that has infiltrated the tooth structure.70,79 fatigue investigations have indicated that the overall time - dependent behavior of the composite tooth interface is a complex function of the individual material phases . for example , microfinite element analyses have shown that each material phase at the a / d interface experiences different stress concentrations at functional loads.96,97 the overall failure behavior of the bond at the a / d interface is not determined by the weakest component but by the component whose stress concentration is closest to its failure strength . similarly , the overall fatigue life of the a / d interface is governed by the material component with the shortest fatigue life under a given loading condition.98 under masticatory function the material components at the composite tooth interface are subjected to both chemical and mechanical stresses . the interplay between these stresses can result in a deterioration of the properties of the material over time . the breaking of covalent bonds by addition of water to ester bonds is considered one of the primary reasons for deterioration of the adhesive at the interface between the composite and tooth.89,90 interestingly , degradation of methacrylate ester groups produces carboxylic acids the same functional group that is the culprit in lactic acid - induced dental caries . the change in mechanical properties of the materials can be contributed to a variety of mechanisms that include proliferation of surface and subsurface flaws.95,97,99101 these flaws in combination with the chemical and biochemical stresses that are present in the mouth can then lead to restoration failure . in conclusion , the a / d bond can be the first defense against substances that may penetrate and ultimately undermine the gingival margin in composite restorations in vivo . it has been hypothesized that the in vivo degradation of the bond at the a / d interface follows a cascade of events that begins when the dentin is acid etched102,103 disruption of the tooth structure by acid etching exposes and activates proteolytic enzymes , eg , matrix metalloproteinases ( mmps ) , that can degrade the exposed collagen component of the hybrid layer.104,105 the following factors inhibit the formation of a durable a / d bond : ( 1 ) water sorption and hydrolysis of the adhesive resin ; ( 2 ) inadequate monomer / polymer conversion of the infiltrating adhesive ; ( 3 ) incomplete resin infiltration of the demineralized dentin matrix ; ( 4 ) incomplete solvent evaporation ; and ( 5 ) enzymatic challenges within the cavity preparation through exposure to oral fluids.49,71,83,104115 although durable a / d bonds are critical for maintaining a seal at the tooth composite interface , the properties of the materials are only one part of an extremely complex problem.116 restoring posterior teeth with resin - based composite materials continues to gain popularity among clinicians , and the demand for such aesthetic restorations is increasing . manufacturers are working aggressively to improve resin composite materials by modifying components to decrease polymerization shrinkage , to improve mechanical and physical properties , and to enhance handling characteristics . the two main causes of posterior composite restoration failure are secondary caries and fracture ( restoration or tooth).35 a review and update of posterior resin composites in terms of preparation design , matrix choice , and resin systems demonstrate the limited extent to which these factors influence the overall clinical lifetime of resins placed in posterior teeth . clinical and patient factors , including caries risk , cavity size , cavity type , number of restored surfaces , and position of the tooth in the mouth must be given careful attention in the selection of any restorative material including composite resins . while clinicians tend to focus on tooth form and function when evaluating the success and failure of posterior resins , the emphasis must remain in advancing our understanding and knowledge of the intricate and complicated characteristics of the restoration tooth interface . this paper presents an update in existing technology and underscores the mechanisms that negatively impact the durability of posterior composites in permanent teeth .

the purpose of our systematic review is to document the adverse events that follow phacoemulsification in eyes with trabeculectomy due to glaucoma and to determine whether phacoemulsification jeopardizes the survival of the trabeculectomy . our research was based on english- and non - english - language articles obtained using the medline , embase , web of science and scopus databases . additional studies were identified by searching bibliographies in the british library and abstracts presented at the association for research in vision and ophthalmology annual meetings . search terms included randomized controlled trial , controlled clinical trial , random allocation , double - blind method , matched studies and trabeculectomy failure , glaucoma - filtering operation failure , bleb failure and cataract surgery or phacoemulsification . only prospective or retrospective matched studies testing the survival of a trabeculectomy alone versus clear corneal phacoemulsification after a trabeculectomy in patients with glaucoma were included . data were independently extracted by two authors using predefined data fields . pubmed yielded 152 results , scopus 235 , embase 222 , and web of science ( science citation index ) 216 . we read the abstracts of all the trials , and after reading the full text of 31 studies , we decided that 13 studies should be comprehensively evaluated . current evidence does not allow us to draw safe conclusions on the scientific question so far . restoring posterior teeth with resin - based composite materials continues to gain popularity among clinicians , and the demand for such aesthetic restorations is increasing . indeed , the most common aesthetic alternative to dental amalgam is resin composite . moderate to large posterior composite restorations , however , have higher failure rates , more recurrent caries , and increased frequency of replacement . investigators across the globe are researching new materials and techniques that will improve the clinical performance , handling characteristics , and mechanical and physical properties of composite resin restorative materials . despite such attention , large to moderate posterior composite restorations continue to have a clinical lifetime that is approximately one - half that of the dental amalgam . while there are numerous recommendations regarding preparation design , restoration placement , and polymerization technique , current research indicates that restoration longevity depends on several variables that may be difficult for the dentist to control . these variables include the patient s caries risk , tooth position , patient habits , number of restored surfaces , the quality of the tooth restoration bond , and the ability of the restorative material to produce a sealed tooth restoration interface . although clinicians tend to focus on tooth form when evaluating the success and failure of posterior composite restorations , the emphasis must remain on advancing our understanding of the clinical variables that impact the formation of a durable seal at the restoration tooth interface . this paper presents an update of existing technology and underscores the mechanisms that negatively impact the durability of posterior composite restorations in permanent teeth .

sarcoidosis is a rare condition with a prevalence of 4.46.3 patients per 100,000 personyears in australia , a figure similar to that of the united state 1 , 2 . it is a multisystem granulomatous disorder of unknown etiology predominantly affecting young and middleaged adults 3 , 4 . diagnostic criteria are based on a compatible clinical and radiological features supported by the histological evidence of noncaseating granulomas in the affected tissues , and the exclusion of other known causes of granulomatous inflammation 3 , 4 . pulmonary infiltration and hilar lymphadenopathy are the most common findings in more than 90% of cases , and transbronchial lung biopsy has been the recommended diagnostic procedure 3 , 4 . other organs involvement includes skin , eyes , liver , spleen , heart , musculoskeletal system , gastrointestinal tract , bone marrow , and the central nervous system . anemia has been reported in cases of sarcoidosis with the frequency ranging from 3.4% to 31% with a variety of explanations . these include hypersplenism , burden of chronic disease , autoimmunity , and bone marrow infiltration 5 . histological evaluation of extrapulmonary sarcoidosis depends on the site of involvement , but its diagnosis is rarely made via bone marrow biopsy 6 . features which may suggest bone marrow infiltration in sarcoidosis include extrapulmonary involvement and varying degree of cytopenia 7 . the characteristic granuloma in sarcoidosis is a noncaseating focal aggregation of macrophages or epithelioid cells , with or without multinucleated giant cells . its presence confirmed the diagnosis of a nonnecrotizing granuloma , in particular sarcoidosis 8 , 9 . a 40yearold caucasian woman presented with a threemonth history of polyuria , malaise , and weight loss . she had been receiving interferon ( ifn ) 1 for multiple sclerosis for the previous 12 years . investigations revealed moderate normocytic anemia , hypercalcemia , hyperglobulinemia , and acute kidney injury ( table 1 ) . her initial chest radiograph was normal , but further investigation with computed tomography showed diffuse lymphadenopathy in her chest and abdomen . a bone marrow trephine biopsy demonstrated noncaseating granulomas ( figs 1 and 2 ) and asteroid bodies ( black arrows in fig . her ifn treatment was discontinued , and she was commenced on prednisolone ( 60 mg daily ) with a slow tapering regimen over 6 months . her renal function and hypercalcemia recovered promptly , and her hemoglobin normalized at 12th week . summary of laboratory investigation results egfr , estimated glomerular filtration rate ; pth , parathyroid hormone ; k / l , kappa / lambda light chain ; ace , angiotensin converting enzyme ; si , systme international . histopathological section of a trephine bone marrow biopsy revealing a noncaseating granuloma composed of aggregation of multinucleated giant cells containing asteroid bodies ( arrowed ) . the noncaseating granuloma in the center ( black arrow ) is surrounded by pale pink amorphous material with scattered mononuclear cells interspersed throughout with some giant cells . there is also a surrounding cuff composed of reactive cells such as eosinophils and neutrophils . a second granuloma is visible in the periphery containing a langhans giant cell ( white arrow ) . a cardinal feature of sarcoidosis is the presence of cd+ t cells which display unregulated interaction with the antigenpresenting cells to initiate the formation and maintenance of noncaseating granulomas 10 . oligoclonal tcell repertoire observed in sarcoidosis suggests that the triggering antigens favor the progressive accumulation and activation of selective cd4 tcell clones 11 . this in turn leads to the preferential differentiation of the th1 helper cells which predominantly secretes interleukins ( il)2 and il12 12 . these ils have been found in higher quantity in the bronchoalveolar lavage fluid of patients with sarcoidosis and subsequently stimulate increased production of ifn and macrophage activation . it has a different structure and binds to a different receptor than the type i ifn ( and ) , and its gene is located on a different chromosome 12 . although endogenous ifn has been implicated in the pathogenesis of sarcoidosis , there is little evidence for other types of ifns 11 . the incidence of sarcoidosis is increasing with the use of type i ifn ( ifn , ifn ) for various conditions such as hepatitis b , hepatitis c , lymphoproliferative malignancy , and multiple sclerosis 13 , 14 , 15 . to date , 60 cases of sarcoidosis have been reported in the english literature in association with the use of ifn , in comparison with only six cases which were associated with the use of ifn 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 . the mean onset of development of sarcoidosis was 11.4 months after initiation of ifn therapy ( ranged 160 months ) , which is much earlier than our patient 13 . the pattern of system involvement is similar to that of the idiopathic form of sarcoidosis . only one case of the six is proven to have bone marrow involvement with a bone marrow biopsy 16 . ifninduced sarcoidosis exhibits a relatively mild disease course that usually resolves with cessation of ifn treatment . a small proportion of patients required systemic corticosteroid 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 . it is a diagnosis that should be considered in patients receiving ifn therapy of any type who present with anemia and multiorgan dysfunction . the overall pattern of organs involvement in sarcoidosis associated with ifn is similar to that of the idiopathic form . apratoxins are potent cytotoxins derived from marine cyanobacteria . because of their biological activity and intriguing structures , we recently established that apratoxins prevent cotranslational translocation and thereby downregulate various receptors , including receptor tyrosine kinases ( rtks ) , and inhibit trafficking of other secretory molecules , including growth factors that act on rtks . rtks such as epidermal growth factor receptors and corresponding ligands such as vascular endothelial growth factor a ( vegf - a ) individually are validated drug targets , which resulted in the approval of small molecules and antibodies against these proteins for colorectal cancer and other cancers . the combined indirect inhibition of both classes of molecules by apratoxins has proven very powerful and an alternative to the specific targeting of selected secretory proteins in cancers that rely on autocrine loops such as colorectal cancer . apratoxin a was rigorously profiled and shown to possess broad - spectrum yet differential in vitro activity ; however , it also showed irreversible toxicity in vivo and was not well tolerated . our initial investigation of the structure activity relationships indicated that the irreversible toxicity may be an off - target effect rather than mechanism - based and may be related to the presence of the michael acceptor in the molecule that might be prone to nonspecific addition of cellular nucleophiles . indeed , in vitro experiments showed that michael addition at c29 of apratoxin a can occur with thiol - containing compounds such as glutathione , cysteine , and n - acetylcysteine ( supporting information , scheme s1 ) . unfortunately , the only natural apratoxin without that ,-unsaturated carbonyl system has been apratoxin e , which , however , exerts much reduced activity , presumably due to the dehydration of the tertiary alcohol at c35 . therefore , we designed and synthesized apratoxin s4 ( 1a ) , which represents a hybrid of the natural products apratoxins a and e , containing the hydroxy group as in apratoxin a that is important for potent activity and lacking the michael acceptor as in apratoxin e ( figure 1 ) . compound 1a showed greater potency and efficacy in a colorectal tumor xenograft model than the parent compound ( apratoxin a ) and was much better tolerated in vivo , providing evidence that the apratoxin scaffold can serve as a new modality to treat cancer but also validating the mechanism of action as a new therapeutic strategy . natural apratoxins a and e and synthetic apratoxin s4 . in our previous work , some fragments of 1a were prepared with modified methods that paralleled those by other groups . to enable the large - scale synthesis of 1a for rigorous preclinical assessment , several steps needed to be improved or replaced due to their low efficiency or harsh operation . an improved synthesis would also provide easier access to additional apratoxin analogues , which we designed based on earlier observations with the goal of improving metabolic stability and reducing complexity of the molecule . in apratoxin a , the chiral center at c34 bears the risk of epimerization ; however , the c34 epimer has the same potency as apratoxin a and the same holds true for 1a and 34-epi-1a ( apratoxin s6 ) . it is also known that the hydroxy group at c35 in apratoxin a is sensitive toward acid - induced dehydration , leading to the double bond between c34 and c35 , and the product has greatly decreased activity , thus presenting a potential major deactivation pathway for 1a as well . on the basis of the above , it was necessary to explore and investigate analogues at c34 : ( 1 ) nonmethyl - c34 , to remove the chiral center , thereby simplifying the molecule ( apratoxin s7 , 1b ) , and ( 2 ) gem - dimethyl - c34 , to remove the chiral center to simplify the molecule and reduce the possibility to dehydrate to form a conjugated system with the thiazoline ( apratoxin s8 , 1c ) . both analogues were expected to retain potent activity based on our previous results with 34-epi-1a . to continue our systematic sar investigation in the nonpeptide portions , we also aimed to explore the synthesis and activity of 30-epi-1a ( apratoxin s9 , 1d ) . here we show the details of an improved synthesis of 1a and the synthesis of analogues with different c34 methylation status ( 1b , c ) and configuration of c30 ( 1d ) . during the development of this improved synthesis , several new reactions were applied , key reactions were modified to meet different substrate requirements , and many steps were optimized , resulting in an efficient synthesis that enables the generation of large - scale quantities of apratoxins for preclinical evaluation . we studied the sar by subjecting all new synthetic apratoxins to a multidimensional assay platform , measuring cell growth , receptor tyrosine kinase receptor ( rtk ) levels , and growth factor secretion . we also investigated the in vitro stability of these compounds and tested the compound with the least propensity to be deactivated via dehydration ( 1c ) for in vivo antitumor efficacy . the main strategies and procedures in the synthesis of all analogues we designed paralleled those of reported procedures but with notable modifications . on the basis of the retrosynthetic analysis depicted in scheme 1 , 1a and its analogues 1b d can be synthesized from two parts : tripeptide 6 and thiazole - containing long chain aliphatic acid 2 . the key step for the synthesis of each analogue is the formation of the thiazoline ring in 2 from the open - chain precursor 3 . experiments below showed that different degrees of methylation at c34 or different configuration of c30 affected the ring formation to different extents . compound 3 can be disconnected into modified cysteine 5 and proline ester based carboxylic acid 4 , which in turn can be synthesized from aldehyde 7 through a series of known reactions . scheme 2 details the steps which were improved from published procedures to meet practical large - scale production . -hydroxy ketone 8 was prepared via d - proline catalyzed aldol reaction of pivalaldehyde with acetone . after protection with tbs to 9 , reduction with nabh4 to 10 , and elimination through mesylate , 8 was transformed into allyl tbs ether 11 . the cleavage of the tbs from 11 afforded allyl alcohol 12 , which is the most critical step in the preparation of 12 due to its volatile property . the solvent ( et2o and thf ) in 12-containing fractions can be removed by distillation using a vigreux fractionation column for small - scale reactions under normal pressure ; however , for large - scale reactions , this was not feasible and we used the combination of cooling - concentration method under ambient reduced pressure ( see supporting information , figure s1 ) and vigreux fraction concentration . even though our reaction sequence consisted of more steps to prepare 12 than brown s method using ipc2bome , the latter caused purification problems which we did not encounter utilizing our optimized method . we found that this is indeed a practical route to the large - scale production of 12 , which we could easily achieve on 10 g scale . according to published procedures with minor modifications , aldehyde 7 was prepared starting from allyl alcohol 12 through formation of acryloyl ester 13 , grubbs catalyst - effected rcm reaction ( 14 ) , methylation with me2(cucn)li2 ( 15 ) , weinreb amide formation ( 16 ) , protection of hydroxy group with pmb ( 17 ) , and reduction with dibal - h ( 7 ) . the yield was low ( 1025% ) when we used p - methoxybenzyl bromide ( pmbbr ) to protect the hydroxy group of 16 in the presence of nah / tetra - n - butyl - ammonium iodide ( tbai ) in thf or nah in dmf . fortunately , we found that the pmboc(nh)ccl3/tfoh method was effective in smoothly converting 16 into pmb ether in moderate yield ( 56% ) . furthermore , unreacted starting material 16 was recovered quantitatively and could be used in the next cycle . it is noteworthy that , to shorten the synthetic route , we also tried to apply a grignard reaction to the syn - cyclic sulfate derivative of 8 to afford aldehyde 7 ( supporting information , scheme s2 ) . using this strategy , 8 should be stereoselectively reduced to the syn - diol . when we performed small - scale reactions ( < 2 g ) , we obtained adequate results ; however , for large - scale reactions ( > 4 g ) , both methods provided unsatisfactory results : low yields , low diastereoselectivity , and most of the product was obtained as an inseparable mixture . we speculate that an extremely slow rate of addition of reducing reagents is crucial for the success of this reaction . with aldehyde 7 in hand , we prepared proline ester - based aliphatic acid 4a c using modified published procedures ( scheme 3 ) . c34-methyl 4a and c34-nonmethyl 4b were synthesized utilizing a similar strategy ( scheme 3a ) . in our previous work , we constructed the c34(me)c35(oh ) chiral unit of 19a through roush s crotylation with roush s ( e)-crotylborate at 78 c in toluene following published procedures . roush s ( e)-crotylborate is not commercially available , it is laborious to purify crude roush s ( e)-crotylborate prepared in the lab by distillation and , if crude product was used without purification , the impurity reduced the yield to as low as 55% . here we now applied leighton s silanes 18a , b instead of roush s crotylborate to construct the c34c35 units in 19a , b . silanes 18a and 18b are commercially available and inexpensive ; they are solid and easy to handle . compound 19a and 19b were obtained smoothly in high yields when aldehyde 7 was treated with 18a and 18b along with sc(otf)3 in ch2cl2 at 0 c , respectively . compound 19a was converted into troc ester 20a with 2,2,2-trichloroethoxycarbonyl chloride ( troccl ) in the presence of pyridine and dmap . the troc ester of 19b was not stable and partly dehydrated to form a conjugate double bond , which led to low yield and caused problems with purification . therefore , we protected hydroxy group 19b with tbs and smoothly obtained the stable tbs ether 20b . the removal of the pmb group and subsequent esterification with fmoc - pro - oh by yamaguchi s method provided prolyl ester 21a and 21b . the combination of oxidants , oso4/oxone and naio4 , was effective to oxidize 21a to carboxylic acid 4a ; however , when 21b was exposed to the same conditions , the tbs group was cleaved simultaneously and gave carboxylic acid 4b with a free hydroxy group . the cleavage of the tbs group probably resulted from the acidic property of oxone which is a triple salt 2khso5khso4k2so4 . both 4a and the enantioselective chiral borane - mediated aldol reaction developed by kiyooka was used to construct the -hydroxy-,-dimethyl acid part of c34-gem - dimethyl acid 4c ( scheme 3b ) . treatment of aldehyde 7 with methyl trimethylsilyl dimethylketene acetal at 78 c in the presence of chiral oxazaborolidinone 22 ( derived from d - val ) provided ( s)--hydroxy ester 23 . with 23 in hand , we first tried to protect the hydroxy with the troc group and then hydrolyze the methyl ester with lioh . however , we found that troco-23 was not compatible to the lioh aq solution during hydrolysis in which a complex mixture including troc - cleaved acid was generated . we planned to couple 24 with modified cysteine ( mocys ) unit 5a first and then protect the hydroxy group with troc and finally remove pmb to form proline ester . however , we found the esterification from this reaction sequence provided a very low yield and caused difficulty in the purification . therefore , we adjusted the reaction sequence to esterify with fmoc - pro - oh first and then install the mocys unit . hydroxy acid 24 was converted into allyl ester 25 with allylbr in the presence of k2co3 in 93% yield . similarly to the preparation of 21a , b , hydroxy ester 25 was protected with troc and the pmb group was removed followed by esterification with fmoc - pro - oh to provide prolyl ester 27 , which was transformed into the carboxylic acid 4c by treatment with pd catalyst and n - methylaniline . the synthesis of modified cysteine unit 5a and its enantiomer 5b was achieved by following published procedures with minor modifications as depicted in scheme 4 . the weinreb amide 28 from s- ( or r- ) n - boc - cys(s - trt)-oh was reduced to amino aldehyde 29 , which was selectively converted into chain - extended modified cysteine ( e)-30 by wittig olefination . during this conversion , the choice of reductant was of importance because -amino aldehydes are easily racemized . the method using lithium aluminum hydride ( lialh4 ) in thf at 0 c decreased the racemization rate compared with that using dibal - h in toluene at 78 c in this preparation . subsequent reduction of the conjugated double bond in 30a and 30b using nabh4 in 95% ethanol afforded saturated compounds 31a and 31b , respectively , but only in 35% yield for both of them . to increase this yield , we also attempted hydrogenation with rhcl(pph3)3/h2 in toluene at 50 c . this reaction , however , gave a similar yield but also posed problems during the chromatographic purification of the product , which was considered a greater problem . fortunately , our apratoxin synthesis is convergent and this low yield is still not detrimental because it is not part of the longest linear sequence . finally , ethyl esters 31 were transformed into allyl ester 5 through hydrolysis and subsequent alkylation . using modified reported procedures,3a d and 2a d were synthesized as depicted in scheme 5 . the n - boc groups in 4a c were selectively removed with tmsotf in the presence of 2,6-lutidine and subsequent coupling of deprotected 4a c with 5a or 5b gave 3a d . first , we screened several coupling reagents for the preparation of 3a ( table 1 , entry 1 ) . even though hatu did not provide a good yield in the preparation of 3a , the yield was as high as 82% when it was used in the preparation of 3b . acknowledging that it may be difficult to prepare 3c due to the steric hindrance of -gem - dimethyl in acid 4c , we also screened coupling reagents for its preparation ( table 1 , entry 2 ) : pyaop gave the highest yield of 89% and surprisingly the bep coupling product gave a complex proton nmr as well as low yield while hatu gave an even lower yield of 20% . considering the similarity of 3d to 3a and the problem with bep to obtain 3c , we used bop as coupling reagent but only obtained product in moderate yield ( 62% ) ; however , pyaop provided a high yield of 91% ( table 1 , entry 3 ) . with 3a d in hand , we synthesized thiazoline - based acids 2a d ( scheme 5 ) . the formation of thiazoline ring is the key step in the preparation 2a d . replicating published procedures using kelly s method , ph3p = o / tf2o induced thiazoline ring formation smoothly for 3a , b to give 33a , b in ch2cl2 at 0 c within 30 min . however , for 3d , it took 3 h to consume all starting material and to finally form cyclized compound 33d . for gem - dimethyl 3c , it was surprising to us that ph3p = o / tf2o was unable to induce thiazoline formation effectively ( table 2 ) , despite extended reaction time or increased reaction temperature . solely the reaction at 60 c for 24 h gave 29% of 33c ( table 2 , entry 5 ) . under other conditions , we only detected trace amounts of product ( table 2 , entries 14 ) . then we turned to kelly s ticl4 method using conditions as shown in table 2 . for ticl4 mediated thiazoline formation , at 2540 c in 540 h , only 3034% yield was obtained ( entries 67 ) ; however , the yield improved to 72% when the reaction temperature was increased to 60 c ( table 2 , entry 8) . thiazoline - containing intermediates 33a d were immediately treated with zn - nh4oac to remove the troc group to avoid elimination of the o - troc group , providing 34a d in 5590% yield . in the preparation of 34a b , the percentage of their dehydration products was : dehyd-34b = 15.5% dehyd-34d trace > the removal of allyl esters of 34a d using pd(pph3)4/n - methylaniline provided acids 2a d . reactions were carried out using ph3p = o ( 8 equiv ) and tf2o ( 4 equiv ) for entries 15 . ticl4 ( 5 equiv ) for entries 68 ( 10 mg scale reactions ) or 2.53.0 equiv ticl4 used for > 30 mg scale reactions ) . this reaction first was carried out at 25 c for 24 h , when large amounts of starting material were still found by tlc and ms , and then this reaction was heated under reflux for another 16 h. reactions were monitored by ms , the bands of starting material and product on tlc were very close . products were isolated using preparative tlc plates . with 2a d in hand , we synthesized the final targets 1a d ( scheme 6 ) . fmoc - protected tripeptide 6(2a2c ) was treated with et2nh in mecn to liberate the corresponding amine and then coupled with acids 2a d to provide 36a d in yields of 7195% . pyaop was chosen as the coupling reagent in the preparation of 36a , c , d with acceptable results . however , for c34-nonmethyl - acid 2b ( table 3 ) , when pyaop or hatu were used as coupling reagents , product 36b was obtained in only low yield ( 1035% ) along with dedydration product ( dehyd-36b ) ( 520% isolated yield ) . one of the reasons for the low yield of 36b was that starting material 2b was consumed by intramolecular dehydration ( via elimination to form a double bond at c34c35 or intramolecular cyclization to form lactone between cooh and c35-oh of 2b ) . when we used depbt , a specific reagent for amine coupling , the desired product 36b was obtained in 72% yield and 13% of dehyd-36b was isolated , while only trace amount of intramolecular dehydration compound of 2b was detected . the cleavage of o - allyl ester by pd(pph3)4/n - methylaniline and the removal of fmoc by et2nh / mecn from linear precursors 36a d gave unmasked reactive precursors , which were then macrocyclized with pyaop or depbt in diluted solution . subsequent purification by semipreparative hplc afforded final targets 1a d in yields of 60% , 25% , 70% , and 45% , respectively . along with 1b and 1d , there were 10% and 5% dehydrated cyclized compounds isolated , respectively . the yields of cyclization and final total yields for the longest linear sequence from pivalaldehyde are summarized in table 4 . the most optimized yield ( 5.0% ) the lower yields of 1b and 1d resulted mainly from their dehydration propensity at c35 during the formation of the thiazoline ring and macrocyclization steps . however , the lower total yield of 1c is due to the lower efficiency of the thiazoline ring formation , presumably as a result of steric hindrance of gem - dimethyl at c34 . nevertheless , the prevention of dehydration of gem - dimethyl in the formation of the thiazoline ring ( 34c ) and the best yield ( 70% ) during macrocyclization ( 1c ) supported the utility and synthetic accessibility of the gem - dimethyl analogue at c34 . based on the reaction sequence : pivalaldehyde 7 4 3 2 1 . compounds 1b d retained potent activity compared with 1a in all biological assays . as hypothesized at the onset of this study and consistent with our previous data that the cytotoxic activity is independent of the configuration of methyl - c34 , we found that the nonmethylated compound ( 1b ) as well as the gem - dimethyl analogue ( 1c ) have similar antiproliferative activity between 1 and 2 nm ( table 5 ) . this indicates that the complexity of apratoxins by eliminating one chiral center ( c34 ) can be somewhat reduced . we have previously shown that apratoxins inhibit cotranslational translocation of secretory molecules , including receptors and growth factors , and therefore we measured representative key members of these protein classes . the low - nanomolar antiproliferative activity is paralleled by a similar potency in reducing levels of met proto - oncogene receptor ( met ) ( figure 2 ) , representative for receptor tyrosine kinases ( rtks ) overexpressed in cancers . we also measured secretion of the angiogenic vegf - a , which is potently inhibited even at subnanomolar concentrations by 1b and 1c with ic50 values of 300 and 470 we had been unable to predict the biological consequences of inverting the c30 configuration of the thiazoline ring . interestingly , the 30-epi-1a , compound 1d , showed superior , picomolar ( subnanomolar ) potency in all three assays , outperforming the other apratoxins by 23-fold ( table 5 , figure 2 ) . determined after 48 h ( n = 4 ) . sar for synthetic apratoxins by immunoblot analysis for rtk ( met ) levels . compounds 1a d were remarkably stable ( t1/2 > 24 h ) under aqueous conditions at physiological ( ph 7.4 ) and lysosomal ph ( 4.88 ) ( figures 3a , b ) and possessed excellent plasma and cellular stability ( figures 3c , d ) . under these conditions , the c34c35 dehydrated and hydrated compounds were probably also not interconverting because we also did not observe any hydration at c28c29 of the conjugated system in apratoxin a. we had been concerned about the tendency of apratoxins a , 1a , 1b , and 1d to dehydrate during synthesis as well as upon prolonged exposure to acidic organic solvents ( e.g. , chloroform ) , which could represent a major deactivation pathway for the apratoxins because the dehydrated compounds possess much reduced activity . while an issue during synthesis ( see above ) and in organic solvents , however , this appeared to not be a major concern in our biological in vitro systems . nevertheless , 1a and 1b were generally somewhat less stable than 1c , as expected ( figure 3 ) , especially under acidic conditions ( figure 3b ) . the stability of 1d was also slightly enhanced compared with that of 1a , suggesting that the configuration at c30 may affect the tendency to dehydrate ( possibly by changing the macrocycle conformation ) , assuming that dehydration is indeed the mode of primary apratoxin modification . in the synthesis , however , the dehydration propensity at c35 during the formation of the thiazoline ring and macrocyclization steps was slightly greater for 1d compared with 1a . microsomal metabolism of all apratoxins was strongly accelerated by nadph and stability was found to be low ( t1/2 < 5 min , table 6 ) , which may suggest that certain apratoxin biotransformation products could also retain activity , considering that 1a was extremely potent and active in vivo as well . in vitro stability of apratoxins under various conditions . apratoxins were incubated as indicated and extracted with ethyl acetate , subjected to lc - ms and monitored by using compound - specific mrm mode with harmine as internal standard . ( d ) cellular stability upon exposure to hct116 protein lysate ( 0.7 mg / ml ) . mean values are shown sd . even though 1c had slightly lower activity than our other synthetic apratoxins , it was still very potent in vitro and had the potential advantage that it can not dehydrate to form a conjugated system . therefore , we tested 1c for efficacy in the same hct116 xenograft mouse model as previously performed for 1a(2a ) and administered 1c at the previous efficacious dose for 1a ( 0.25 mg / kg , 5 g / mouse ) and a 2.5-fold lower dose ( 0.1 mg / kg , 2 g / mouse ) daily via intraperitoneal ( ip ) injection for 16 days . there was a clear dose - dependent tumor growth inhibition ( figure 4a ) . the higher dose strongly retarded the tumor growth , paralleling the effects of 1a . the lower dose showed a weaker yet pronounced effect ( figure 4a ) ; however , the tumor growth could only be inhibited by about 50% using 0.1 mg / kg . it is noteworthy that we did not observe signs of toxicity based on lack of both weight loss and abnormal behavior . although vegfr2 expression in hct116 xenografts was found to be low ( consistent with literature data ) , at the higher dose of 1c , there was a detectable reduction in vegfr2 expression levels , indicating target engagement in vivo ( figure 4b ) . dose - dependent in vivo activity of 1c using a hct116 xenograft mouse model . ( a ) subcutaneous tumor - bearing mice were injected daily ip with 1c ( n = 7 ) or vehicle ( n = 6 ) , and error bars indicate sem ( b ) at the end of the efficacy study , tumors were analyzed by immunoblot analysis for levels of an rtk . apratoxin s4 ( 1a ) along with its hitherto unknown c30 epimer ( 1d ) and two analogues with achiral c34 ( 1b , c ) were synthesized using improved procedures . the innovative points in the synthesis are : ( 1 ) preparation of homoallylic alcohol 12 from its tbs ether directly working up by cooling - concentration , which is critical to the scale up of aldehyde 7 , ( 2 ) leighton s silanes were introduced to efficiently prepare -hydroxy acid 4 , one key segment of composing apratoxins , ( 3 ) lialh4 was used instead of dibal - h to prepare amino aldehyde in the preparation of mocys part to greatly decrease the racemization , ( 4 ) kelly s thiazoline formation methods ( both ph3p = o / tf2o and ticl4 ) were developed to meet different substrate requirements , and ( 5 ) the optimized steps are efficient for the synthesis of 1a and viable to generate 1c and 1d . as hypothesized , sar studies demonstrated that 1b and 1c retain activity compared with 1a . unexpectedly , we identified 1d as the most potent apratoxin to date , exhibiting 23-fold improved in vitro activity , even though 1d still has the potential to be deactivated by dehydration . however , compound 1d was found to be stable during repurifications and stability tests . the propensity of apratoxins to dehydrate to form a c34c35 double bond is decreased with each methylation at c34 ; this is possibly a reason why the methylene - c34 has not been isolated from cyanobacteria but only the dehydrated form , apratoxin e ( figure 1 ) , which could have been formed enzymatically or nonenzymatically . while dehydration appears to not be a problem during in vitro incubations and stability studies , it may lead to apratoxin deactivation in vivo so that the corresponding gem - dimethyl compound 1c was further characterized and shown to possess in vivo antitumor efficacy . the low microsomal stability combined with the potent in vivo activity may suggest that apratoxin biotransformation products could play a role in the overall activity , and future experiments are aimed at characterizing the in vivo biotransformation products . overall , our study further corroborates that the development of this class of anticancer agents is warranted . tetrahydrofuran ( thf ) and diethyl ether ( et2o ) were distilled from sodium chips in the presence of a small amount of benzophenone ; ch2cl2 and toluene were distilled from cah2 ; mecn , n , n - dimethylformamide ( dmf ) were dried with 4 molecular sieves ( ms ) and meoh dried with 3 ms ; 4 m hydrochloric acid ( hcl ) solution in ethyl acetate was prepared by dissolving hcl gas ( yielding by dropping aqueous hydrochloric acid ( 34% ) to concentrated sulfuric acid ( 98% ) ) to ethyl acetate . all reactions were performed in heat - gun dried flasks ( 400 c under reduced pressure ) under an inert atmosphere of anhydrous ar unless otherwise noted . thin layer chromatography was performed on emd silica gel 60 f254 glass plates and preparative thin layer chromatography was performed on whatman silica gel 60 f254 glass plates ( layer thick 1000 m ) . nuclear magnetic resonance ( nmr ) spectra were recorded on a varian mercury 400 mhz , bruker avance ii 600 , bruker avance iii 600 mhz , or agilent vnmr 600 mhz spectrometer as indicated in the data list . chemical shifts for proton nuclear magnetic resonance ( h nmr ) spectra are reported in parts per million relative to the signal residual cdcl3 at 7.26 ppm . chemicals shifts for carbon nuclear magnetic resonance ( c nmr ) spectra are reported in parts per million relative to the center line of the cdcl3 triplet at 77.16 ppm . the abbreviations s , d , dd , ddd , dddd , t , q , br , and m stand for the resonance multiplicity singlet , doublet , doublet of doublets , doublet of doublet of doublets , doublet of doublet of doublet of doublets , triplet , quartet , broad and multiplet , respectively . optical rotation was measured on a perkin - elmer 341 polarimeter ( na d line ) using a microcell of 1 dm path length . high resolution mass spectra ( hrms ) data were obtained using an agilent - lc - tof mass spectrometer with an apci / esi multimode ion source detector . lr - ms data was obtained using a 3200 qtrap triple quadrupole mass spectrometer and detection by electrospray ionization - ms in the positive ion mode . the purity ( > 95% ) of all tested compounds was determined by hplc analysis ( shimadzu ) equipped with a phenomenex ultracarb ods column ( 250 mm 10 mm , 5 m ) and a shimadzu spd - m20a detector at 200/220 nm wavelength . the solution of tetra - n - butylammonium floride trihydrate ( tbaf ) ( 82.07 g , 260.116 mmol ) in thf ( 220 ml ) was added to the mixture of compound 11 ( 21.0 g , 86.705 mmol ) and 4 molecular sieves ( 22 g , predried at 450 c under reduced pressure 1 h ) in anhydrous thf ( 300 ml ) at 0 c . then the reaction mixture was stirred at room temperature overnight and filtered through a small pad of celite ( washed with diethyl ether ) . the filtrate was quenched with 200 ml of water , extracted with diethyl ether ( 300 ml 3 ) , washed with brine ( 200 ml 2 ) , dried over anhydrous mgso4 , concentrated with cooling / condensing fraction distillation system under moderate vacuum , and further concentrated by vigreux fraction distillation column . the concentrated mixture was purified by column chromatography eluted by 35% diethyl ether in pentane . the eluted product fractions were also concentrated by cooling / condensing fraction distillation system under moderate vacuum to give product 12 and further distilled by vigreux fraction distillation column to provide product 12 ( 10.5 g , 95% ) . h nmr ( 400 mhz , cdcl3 ) : 5.86 ( dddd , j = 14.4 , 10.4 , 8.8 , 6.0 hz , 1h ) , 5.14 ( m , 2h ) , 3.25 ( dd , j = 10.4 , 2.0 hz , 1h ) , 2.392.33 ( m , 1h ) , 1.98 ( ddd , j = 13.6 , 9.6 , 9.6 hz , 1h ) , 0.91 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 136.7 , 117.9 , 78.2 , 36.7 , 34.7 , 25.9 ppm . 4-methoxybenzyl-2,2,2-trichloroacetimidate ( 3.7 ml , 17.8 mmol ) and trifluoromethane sulfonic acid ( tfoh ) ( 7.9 l , 0.089 mmol ) was added sequentially to the solution of 16 ( 2.05 g , 8.9 mmol ) in thf ( 20 ml ) at 0 c . the resulting mixture was stirred at room temperature overnight and was then diluted with ethyl acetate ( 20 ml ) , quenched with saturated nahco3 ( 20 ml ) , extracted with ethyl acetate ( 20 ml 3 ) , dried with anhydrous mgso4 , and evaporated in vacuo . hexane ( 100 ml ) was added to the residue , which resulted in the precipitation of a white solid ( 2,2,2-trichloroacetimidate ) . the solid was filtered off , and the filtrate was concentrated and purified by column chromatography ( eluted by 2050% ethyl acetate in hexane ) to give product 17 ( 1.74 g , 56% , 100% brsm ) ( recovered starting material 1.08 g and used in next cycle ) . to the solution of 7 ( 234.1 mg , 0.801 mmol ) in ch2cl2 ( 8 ml ) was added ( s , s)-trans ez - crotylmix ( mixture of 18a and sc(otf)3 ) ( 943 mg , 1.602 mmol ) at room temperature . this mixture was stirred vigorously at the same temperature for 2.5 h , and then it was treated with 12 ml of et2o and 12 ml of 1n aq hcl . after this quenched mixture was stirred at room temperature for 30 min , it was filtered off to remove precipitated solid , extracted with et2o ( 15 ml 3 ) , washed with saturated nahco3 ( 20 ml 2 ) and brine ( 20 ml ) , dried with anhydrous mgso4 , evaporated in vacuo , and purified by chromatography column ( eluted by 3.54.5% ethyl acetate in hexane ) to give product 19a ( 245.7 mg , 88% ) as a colorless oil . to the solution of 7 ( 203.3 mg , 0.696 mmol ) in ch2cl2 ( 7 ml ) was added ( s , s)-18b ( 772.1 mg , 1.392 mmol ) and sc(otf)3 ( 28.5 mg , 0.058 mmol ) at 0 c . this mixture was stirred vigorously at the same temperature for 2.0 h , and then it was treated with 12 ml of et2o and 12 ml of 1n aq hcl . afterwards , the quenched mixture was stirred at room temperature for 30 min , filtered to remove the precipitate , extracted with et2o ( 15 ml 3 ) , washed with saturated nahco3 ( 20 ml 2 ) and brine ( 20 ml ) , dried with anhydrous mgso4 , evaporated in vacuo , and purified by column chromatography ( eluted by 4.2% ethyl acetate in hexane ) to give product 19b ( 197.6 mg , 85% ) as a colorless oil ; [ ]d 57.8 ( c 0.32 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 ) : 7.30 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.4 hz , 2h ) , 5.895.79 ( m , 1h ) , 5.175.13 ( m , 2h),4.63 ( d , j = 10.4 hz , 1h ) , 4.52 ( d , j = 10.4 , 1h ) , 3.79 ( s , 3h ) , 3.793.74 ( m , 1h ) , 3.11 ( dd , j = 9.2 , 2.4 hz , 1h ) , 2.302.24 ( m , 1h ) , 2.212.14 ( m , 1h ) , 2.001.90 ( br m , 1h ) , 1.65 ( ddd , j = 13.6 , 10.6 , 2.8 hz , 1h ) , 1.46 ( ddd , j = 14.0 , 8.8 , 4.0 hz , 1h ) , 1.35 ( ddd , j = 14.0 , 9.2 , 2.4 hz , 1h ) , 1.09 ( ddd , j = 13.6 , 10.4 , 2.0 hz , 1h ) , 0.97 ( d , j = 6.4 hz , 3h ) , 0.94 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 159.0 , 135.0 , 131.7 , 129.2 , 118.1 , 113.7 , 85.3 , 74.3 , 68.4 , 55.3 , 43.5 , 43.3 , 39.8 , 36.2 , 26.6 , 21.1 ppm . hrms ( esi ) m / z calcd for c21h34o3na ( m + na ) 357.2400 , found 357.2409 . to the solution of 19b ( 178.6 mg , 0.534 mmol ) in ch2cl2 ( 8 ml ) were added 2,6-lutidine ( 310 l , 2.672 mmol ) and tert - butyldimethylsilyl trifluoromethanesulfonate ( tbsotf ) ( 368.2 l , 1.603 mmol ) at 0 c under ar . after being stirred at the same temperature for 1.5 h , the reaction was quenched with meoh ( 5 ml ) and saturated aq nh4cl ( 7 ml ) , extracted with ethyl acetate ( 10 ml 4 ) , washed with brine ( 10 ml 2 ) , dried with anhydrous mgso4 , evaporated in vacuo , and purified by column chromatography ( eluted by 3.6% ethyl acetate in hexane ) to give product 20b ( 224.4 mg , 94% ) as a colorless oil ; [ ]d 49.0 ( c 0.31 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 ) : 7.28 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.8 hz , 2h ) , 5.865.76 ( m , 1h ) , 5.075.03 ( m , 2h ) , 4.65 ( d , j = 10.8 hz , 1h ) , 4.45 ( d , j = 10.8 hz , 1h ) , 3.863.80 ( m , 1h ) , 3.80 ( s , 3h ) , 3.08 ( dd , j = 7.2 , 3.6 hz , 1h ) , 2.312.20 ( m , 2h ) , 1.87 ( br m , 1h ) , 1.66 ( ddd , j = 13.2 , 9.2 , 2.8 hz , 1h),1.461.34 ( m , 2h ) , 1.06 ( d , j = 6.8 hz , 3h ) , 0.91 ( s , 9h ) , 0.86 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 159.0 , 135.2 , 131.9 , 128.9 , 117.0 , 113.7 , 85.6 , 73.9 , 69.8 , 55.4 , 43.9 , 43.4 , 40.5 , 36.2 , 26.8 , 26.6 , 26.1 , 21.0 , 18.2 , 4.1 , 4.3 ppm . hrms ( esi ) m / z calcd for c27h48o3sina ( m + na ) 471.3265 , found 471.3264 . to a solution of 20 ( 0.806 mmol ) in the mixture of ch2cl2 ( 5.0 ml ) and h2o ( 0.5 ml ) was added 2,3-dichloro-5,6-dibenzoquinone ( ddq ) ( 220 mg , 0.968 mmol ) at 0 c . the reaction mixture was stirred at the same temperature for 1 h , quenched with saturated aqueous nahco3 ( 10 ml ) , and filtered in vacuo . the organic layer was separated and the water layer was extracted with ch2cl2 ( 30 ml 3 ) . the organic phase was combined and washed with brine ( 20 ml 2 ) , dried with anhydrous mgso4 , filtered , and concentrated in vacuo . to the suspended solution of fmoc - pro - oh ( 549.5 mg , 1.629 mmol ) in toluene ( 5.0 ml ) were added n , n - diisopropylethylamine ( diea ) ( 435 l , 2.5 mmol ) and 2,4,6-trichlorobenzoyl chloride ( 390 l , 2.5 mmol ) at room temperature under argon , and the mixture was stirred at the same temperature for 30 min . then the crude alcohol in toluene ( 3.0 ml ) and dmap ( 350 mg , 2.862 mmol ) were added to the above mixture at 10 c . after being stirred at room temperature for 3 h , the reaction mixture was quenched and extracted with diethyl ether ( 10 ml 3 ) . the combined organic layer was washed with saturated nh4cl ( 15 ml 2 ) , saturated nahco3 ( 15 ml 2 ) , and brine ( 15 ml ) , dried over mgso4 , and concentrated in vacuo . the residue was purified by silica gel column chromatography ( eluted by 10% ethyl acetate in hexane ) to give ester 21 . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.787.75 ( m,2 h ) , 7.677.55 ( m , 2h ) , 7.427.38 ( m , 2h ) , 7.337.30 ( m , 2h ) , 5.855.71 ( m , 1h ) , 5.054.99 ( m , 2h ) , 4.80 ( dd , j = 8.4 , 2.0 hz , 1h ) , 4.534.40 ( m , 2h ) , 4.354.17 ( m , 2h ) , 3.813.71 ( m , 1h ) , 3.703.62 ( m , 1h ) , 3.603.50 ( m , 1h ) , 2.322.15 ( m , 3h ) , 2.141.93 ( m , 3h ) , 1.621.49 ( m , 3h ) , 1.391.30 ( m , 1h ) , 1.201.03 ( m , 1h ) , 0.95 ( d , j = 6.4 hz , 2h ) , 0.87 ( s , 9 h ) , 0.87 ( s , 9 h ) , 0.82 ( d , j = 6.4 hz , 1h ) , 0.050.02 ( m , 6h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 172.2 , 172.1 , 154.7 , 154.5 , 144.3 , 144.2 , 144.1 , 143.8 , 141.4 , 141.4 , 141.3 , 135.6 , 135.3 , 127.8 , 127.8 , 127.2 , 127.1 , 127.1 , 125.5 , 125.3 , 125.2 , 120.0 , 116.9 , 116.8 , 80.6 , 80.4 , 77.5 , 77.2 , 76.8 , 70.2 , 70.0 , 67.8 , 67.5 , 59.8 , 59.6 , 47.4 , 47.0 , 46.4 , 44.2 , 44.0 , 42.6 , 42.5 , 39.3 , 39.1 , 35.2 , 35.1 , 31.3 , 30.1 , 27.1 , 27.0 , 26.1 , 26.0 , 26.0 , 24.4 , 23.4 , 20.9 , 20.6 , 18.2 ppm . hrms ( esi ) m / z calcd for c39h57no5sina ( m + na ) 670.3898 , found 670.3920 . to the solution of 21 ( 0.646 mmol ) in dmf ( 6.0 ml ) were added oxone ( 1.588 g , 2.583 mmol ) , nahco3 ( 217.0 mg , 2.583 mmol ) , and oso4 ( 2.5% solution in tert - buoh ) ( 81 l , 6.5 mol ) at room temperature . after being stirred at the same temperature for 15 h , the reaction mixture was diluted with water ( 4 ml ) and tert - buoh ( 7.5 ml ) , and then naio4 ( 276.3 mg , 1.292 mmol ) was added . the reaction mixture was stirred at room temperature for an additional 5 h and poured into aqueous hcl ( 1 m for 4a , 0.5 m for 4b to ph 1 ; 25 ml ) and ch2cl2 ( 25 ml ) . the water layer was extracted with ch2cl2 ( 50 ml 3 ) . the combined ch2cl2 layer was washed with 10 wt % na2s2o3 ( 50 ml 3 ) and brine ( 50 ml 1 ) , dried over mgso4 , and concentrated in vacuo . the residue was purified by silica gel column chromatography to give product 4 as a white solid ( 4a eluted by 1725% ethyl acetate in hexane ; 4b eluted by 2.510% meoh in ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers , major ) : 7.75 ( d , j = 7.6 hz , 2h ) , 7.627.55 ( m , 2h ) , 7.39 ( dd , j = 7.6 , 7.4 hz , 2h ) , 7.337.29 ( m , 2h ) , 5.40 ( br , 1h ) , 4.91 ( d , j = 10.8 hz , 1h ) , 4.424.38 ( m , 1h ) , 4.344.29 ( m , 1h ) , 4.124.06 ( m , 1h ) , 3.683.62 ( m , 1h ) , 3.523.47 ( m , 1h ) , 2.442.43 ( m , 2h ) , 2.332.23 ( m , 1h ) , 2.081.91 ( m , 3h ) , 1.861.80 ( m , 1h ) , 1.75 ( dd , j = 13.6 , 2.4 hz , 1h ) , 1.66 ( dd , j = 14.4 , 2.0 hz , 1h ) , 1.331.26 ( m , 1h ) , 1.040.98 ( m , 1h ) , 0.98 ( d , j = 6.4 hz , 3h ) , 0.89 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers , major ) : 174.6 , 172.4,155.4 , 144.0 , 143.7 , 127.9 , 127.8 , 127.3 , 127.2 , 125.3 , 125.2 , 120.1 , 120.1 , 78.5 , 68.0 , 66.0 , 59.6 , 47.2 , 46.7 , 42.7 , 41.7 , 37.2 , 34.8 , 30.0 , 26.1 , 25.0 , 24.6 , 20.6 ppm . hrms ( esi ) m / z calcd for c32h41no7na ( m + na ) 574.2775 , found 574.2970 . to the solution of n - tosyl - d - val - oh ( 145.6 mg , 0.537 mmol ) in ch2cl2 ( 7 ml ) at 0 c under argon was added the solution of bh3 in thf ( 1 m , 537 l , 0.537 mmol ) dropwise . the resulting mixture was stirred at room temperature for 20 min and then cooled to 78 c . aldehyde 7 ( 157.0 mg , 0.537 mmol ) and methyl trimethylsilyl dimethylketene acetal ( 120.0 l , 0.591 mmol ) were added successively to the above mixture at 78 c . after being stirred at 78 c for 3 h , the reaction mixture was quenched with buffer ( ph 7 ; 8 ml ) and then it was warmed to room temperature and another 5 ml of buffer ( ph 7 ) was added . the quenched reaction mixture was extracted with et2o ( 25 ml 3 ) , washed with saturated nahco3 ( 15 ml 2 ) and brine ( 15 ml 2 ) , dried with anhydrous mgso4 , and purified by column chromatography ( eluted by 8.5% ethyl acetate in hexane ) to give product 23 as a colorless oil ( 171.5 mg , 81% ) ; [ ]d 55.8 ( c 0.12 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 ) : 7.29 ( d , j = 8.4 hz , 2h ) , 6.85 ( d , j = 8.4 hz , 2h ) , 4.63 ( d , j = 10.4 hz , 1h ) , 4.50 ( d , j = 10.4 hz , 1h ) , 3.77 ( s , 3h ) , 3.74 ( br m , 1h ) , 3.69 ( s , 3h ) , 3.09 ( dd , j = 9.2 , 2.4 hz , 1h ) , 2.47 ( br , 1h ) , 2.031.95 ( br m , 1h ) , 1.581.45 ( m , 2h ) , 1.371.30 ( m , 1h ) , 1.18 ( s , 6h ) , 1.031.00 ( m , 1h ) , 0.96 ( d , j = 6.4 hz , 3h ) , 0.93 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 178.3 , 159.0 , 131.6 , 129.2 , 113.7 , 85.2 , 74.4 , 74.2 , 55.3 , 51.9 , 47.3 , 39.7 , 38.2 , 36.2 , 26.8 , 26.6 , 22.1 , 20.9 , 20.6 ppm . hrms ( esi ) m / z calcd for c23h38o5na ( m + na ) 417.2611 , found 417.2628 . to the solution of 23 ( 223.8 mg , 0.568 mmol ) in the mixture of thf meoh h2o ( 7 ml3.5 ml0.7 ml ) was added aq lioh ( 119.1 mg in 2.8 ml h2o , 2.838 mmol ) . after being stirred at room temperature for 5 h , the reaction mixture was diluted with 10 ml of water , acidified by addition of aq hcl ( 2 m ) to ph 2 , extracted with ethyl acetate ( 10 ml 4 ) , washed with brine ( 10 ml 2 ) , dried with anhydrous mgso4 , and purified by column chromatography ( eluted by 8.517% acetone in hexane ) to give 24 ( 225 mg , 100% ) as a white solid ; [ ]d 52.3 ( c 0.26 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 ) : 7.30 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.4 hz , 2h ) , 4.63 ( d , j = 10.4 hz , 1h ) , 4.53 ( d , j = 10.4 hz , 1h ) , 3.80 ( d , j = 13.0 , 1h ) , 3.78 ( s , 3h),3.13 ( dd , j = 8.0 , 2.0 hz , 1h ) , 1.80 ( br m , 1h ) , 1.621.49 ( m , 2h ) , 1.401.34 ( m , 1h ) , 1.21 ( s , 3h ) , 1.19 ( s , 3h ) , 1.09 ( dd , j = 12.4 , 12.4 ) , 0.98 ( d , j = 6.4 hz , 3h ) , 0.95 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 182.9 , 159.0 , 131.4 , 129.3 , 113.7 , 85.2 , 74.3 , 74.3 , 55.3 , 47.1 , 39.4 , 37.9 , 36.2 , 26.8 , 26.6 , 22.3 , 20.8 , 20.2 ppm . hrms ( esi ) m / z calcd for c22h36o5na ( m + na ) 403.2455 , found 403.2472 . to the solution of compound 24 ( 222.4 mg , 0.585 mmol ) in dimethyl sulfoxide ( dmso ) ( 5.0 ml ) were added k2co3 ( 242.5 mg , 1.754 mmol ) and allyl bromide ( allylbr ) ( 99 l , 1.170 mmol ) at room temperature . after being stirred at the same temperature overnight , the reaction was quenched with water ( 10 ml ) and extracted with ethyl acetate ( 20 ml 4 ) . the combined organic layer was washed with brine ( 10 ml 5 ) , filtered , concentrated in vacuo , and purified by silica gel column chromatography ( eluted by 6.5% ethyl acetate in hexane ) to give product 25 ( 228.6 mg , 93% ) as a colorless oil ; [ ]d 56.0 ( c 0.175 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 ) : 7.30 ( d , j = 8.4 hz , 2h ) , 6.85 ( d , j = 8.4 hz , 2h ) , 5.965.86 ( m , 1h ) , 5.355.22 ( m , 2h ) , 4.64 ( d , j = 10.4 hz , 1h ) , 4.60 ( d , j = 5.6 hz , 2h ) , 4.50 ( d , j = 10.4 hz , 1h ) , 3.78 ( br m , 1h ) , 3.77 ( s , 3h ) , 3.10 ( dd , j = 8.0 , 2.0 hz , 1h ) , 2.54 ( br , 1h ) , 2.0 ( br m , 1h ) , 1.611.55 ( m , h ) , 1.49 ( ddd , j = 17.6 , 9.6 , 4.0 hz , 1h ) , 1.371.31 ( m , 1h ) , 1.21 ( s , 6h ) , 1.050.98 ( m , 1h ) , 0.97 ( d , j = 6.8 hz , 3h ) , 0.94 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 177.3 , 158.9 , 132.0 , 131.5 , 129.1 , 118.2 , 113.6 , 85.0 , 74.3 , 74.2 , 65.2 , 55.2 , 47.3 , 39.7 , 38.1 , 36.1 , 26.6 , 26.5 , 21.9 , 20.9 , 20.7 ppm . hrms ( esi ) m / z calcd for c25h40o5na ( m + na ) 443.2768 , found 443.2769 . to the solution of 25 ( 220.0 mg , 0.523 mmol ) in ch2cl2 ( 5.0 ml ) at 0 c under ar were added 4-dimethylaminopyridine ( dmap ) ( 1.3 mg , 10.5 mol ) , pyridine ( 423 l , 5.234 mmol ) , and 2,2,2-trichloroethoxylcarbonyl chloride ( troc - cl ) ( 554.5 mg/360.0 l , 2.617 mmol ) . after being stirred at 0 c for 10 min and room temperature for 1 h , the reaction was quenched with water ( 10 ml ) and extracted with ethyl acetate ( 15 ml 4 ) . the combined organic layer was washed with saturated nahco3 and brine , dried with mgso4 , concentrated in vacuo , and purified by silica gel column chromatography ( 2.51015% ethyl acetate in hexane ) to give 26 ( 295.1 mg , 95.0% ) ; [ ]d 7.6 ( c 0.37 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 ) : 7.27 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.4 hz , 2h ) , 5.965.86 ( m , 1h ) , 5.375.23 ( m , 3h ) , 4.78 ( d , j = 12.0 hz , 1h ) , 4.59 ( d , j = 5.6 hz , 2h ) , 4.55 ( d , j = 10.8 hz , 1h ) , 4.49 ( d , j = 10.8 hz , 1h ) , 4.30 ( d , j = 12.0 hz , 1h ) , 3.78 ( s , 3h ) , 3.05 ( dd , j = 9.6 , 2.0 hz , 1h),1.991.93 ( m , 1h ) , 1.75 ( br m , 1h ) , 1.50 ( ddd , j = 17.2 , 9.2 , 3.0 hz , 1h ) , 1.351.29 ( m , 1h ) , 1.27 ( s , 3h ) , 1.25 ( s , 3h ) , 1.081.01 ( m , 4h ) , 0.92 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 174.8 , 159.0 , 154.4 , 131.9 , 131.5 , 128.7 , 118.6 , 113.7 , 94.6 , 85.0 , 80.9,76.6 , 74.4 , 65.6 , 55.3 , 47.0 , 39.8 , 36.8 , 36.1 , 26.5 , 26.4 , 22.4 , 20.8 , 19.7 ppm . hrms ( esi ) m / z calcd for c28h41cl3o7na ( m + na ) 617.1810 , found 617.1832 . to a solution of 26 ( 291.5 mg , 0.491 mmol ) in a mixture of ch2cl2 ( 5.0 ml ) and h2o ( 0.5 ml ) was added 2,3-dichloro-5,6-dibenzoquinone ( ddq ) ( 133.6 mg , 0.589 mmol ) at 0 c . the reaction mixture was stirred at the same temperature for 1 h , quenched with saturated aqueous nahco3 ( 6 ml ) , and filtered in vacuo . the organic layer was separated , and the water layer was extracted with ch2cl2 ( 15 ml 4 ) . the organic phase was combined and washed with brine ( 3 15 ml ) , dried with anhydrous mgso4 , filtered , and concentrated in vacuo . this residue was used for the next reaction without further purification . to the suspended solution of fmoc - pro - oh ( 334.3 mg , 0.991 mmol ) in toluene ( 3.0 ml ) were added n , n - diisopropylethylamine ( diea ) ( 0.26 ml , 1.49 mmol ) and 2,4,6-trichlorobenzoyl chloride ( 233 l , 1.491 mmol ) at room temperature under argon , which was stirred at the same temperature for 10 min . then the crude alcohol in toluene ( 1.5 ml ) and dmap ( 212.8 mg , 1.742 mmol ) were added to the above mixture . after being stirred at room temperature overnight , the reaction mixture was quenched with water and extracted with diethyl ether ( 20 ml 4 ) . the combined organic layer was washed with saturated nh4cl ( 20 ml 2 ) , saturated nahco3 ( 20 ml 2 ) , and brine ( 20 ml ) , dried over mgso4 , and concentrated in vacuo . the residue was purified by silica gel column chromatography ( eluted by 10% ethyl acetate in hexane ) to give ester 27 ( 360.8 mg , 90.4% ) as a colorless oil ; [ ]d 58.9 ( c 0.124 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.777.75 ( m , 2h ) , 7.677.62 ( m , 2h ) , 7.40 ( dd , j = 7.4 , 7.2 hz , 2h ) , 7.347.29 ( m , 2h ) , 6.005.79 ( m , 1h ) , 5.345.12 ( m , 3h ) , 4.97 ( d , j = 12.0 hz , 0.6h ) , 4.87 ( d , j = 12.0 hz , 0.4h ) , 4.78 ( d , j = 10.0 hz , 1h ) , 4.69 ( d , j = 12.0 hz , 0.4h ) , 4.64 ( d , j = 12.0 hz , 0.6h ) , 4.594.16 ( m , 6h ) , 3.673.61 ( m , 1h ) , 3.593.48 ( m , 1h ) , 2.382.28 ( m , 0.6h ) , 2.242.16 ( m , 0.4h ) , 2.152.06 ( m , 1h ) , 2.041.93 ( m , 2h ) , 1.921.80 ( m , 1h ) , 1.551.48 ( m , 1h ) , 1.461.42 ( m , 1h ) , 1.381.29 ( m , 1h ) , 1.251.19 ( m , 6h ) , 1.151.07 ( m , 0.6h ) , 1.000.96 ( m , 1.6h ) , 0.89 ( s , 5.4h ) , 0.86 ( s , 3.6h ) , 0.67(d , j = 6.4 hz , 1.8h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 175.0,175.0 , 172.9 , 172.4 , 154.7 , 154.5 , 154.3 , 154.2 , 144.4 , 144.3 , 144.0 , 143.9 , 141.4 , 141.4 , 141.3 , 141.3 , 132.0 , 131.9 , 127.8 , 127.2 , 127.1 , 125.5 , 125.4 , 125.3 , 125.2 , 120.0 , 120.0 , 118.6 , 94.9 , 94.8 , 80.8 , 80.6 , 79.8 , 79.5 , 77.1 , 76.9 , 68.0 , 67.5 , 65.7 , 65.6 , 59.9 , 59.4 , 47.3 , 47.3 , 47.2 , 47.1 , 46.5 , 38.2 , 38.0 , 37.3 , 36.8 , 34.9 , 34.8 , 34.7 , 31.7 , 31.4 , 30.1 , 26.9 , 26.7 , 25.9 , 25.9 , 25.4 , 23.5 , 22.8 , 22.0 , 22.0 , 20.5 , 20.4 , 19.8 , 19.7 , 14.2 ppm . hrms ( esi ) m / z calcd for c40h50cl3no9na ( m + na ) 816.2443 , found 816.2420 . to a solution of 27 ( 349.3 mg , 0.440 mmol ) in thf ( 8 ml ) were added pd(pph3)4 ( 76.3 mg , 0.066 mmol ) and n - methyl aniline ( 144.3 l , 1.321 mmol ) at room temperature under argon . this reaction was protected with aluminum foil . after being stirred at room temperature for 1 h , the reaction mixture was concentrated in vacuo and purified by column chromatography ( eluted by acetone / hexane 1:3 ) to give acid 4c ( 354.1 mg , 95% ) as a pale - yellow solid ; [ ]d 51.1 ( c 0.131 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 9.90 ( br , 1h ) , 7.777.74 ( m , 2h ) , 7.687.63 ( m , 2h ) , 7.427.38 ( m , 2h ) , 7.347.29 ( m , 2h ) , 5.23 ( d , j = 10.4 hz , 0.4h ) , 5.13 ( d , j = 10.4 hz , 0.6h ) , 5.01 ( d , j = 12.0 hz , 0.6h ) , 4.90 ( d , j = 12.0 hz , 0.4h ) , 4.854.79 ( m , 1h ) , 4.70 ( d , j = 12.0 hz , 0.4h ) , 4.63 ( d , j = 12.0 hz , 0.6h ) , 4.524.17 ( m , 4h ) , 3.673.48 ( m , 1h ) , 2.372.27 ( m , 0.6h ) , 2.242.19 ( m , 0.4h ) , 2.162.10 ( m , 1h ) , 2.011.83 ( m , 3h ) , 1.781.69 ( m , 0.4h ) , 1.621.49 ( m , 1.6h ) , 1.431.35 ( m , 1h ) , 1.311.20 ( m , 6h ) , 1.091.00 ( m , 1h ) , 1.01 ( d , j = 6.4 hz , 1.2 h ) , 0.94 ( s , 5.4h ) , 0.87 ( s , 3.6h ) , 0.69 ( d , j = 6.4 hz , 1.8h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 181.1 , 180.7 , 172.8 , 172.3 , 154.8 , 154.4 , 154.2 , 144.3 , 144.1 , 143.9 , 143.7 , 141.3 , 141.3 , 141.2 , 141.2 , 135.1 , 135.0 , 135.0 , 130.6 , 128.1 , 128.1 , 128.0 , 127.7 , 127.1 , 127.1 , 127.0 , 125.5 , 125.3 , 125.2 , 125.1 , 120.0 , 119.9 , 94.9 , 94.8 , 80.5 , 80.3 , 79.8 , 79.4 , 77.0 , 76.8 , 68.0 , 67.6 , 59.8 , 59.3 , 47.2 , 47.1 , 46.9 , 46.4 , 38.1 , 37.5 , 37.1 , 36.7 , 34.9 , 34.7 , 34.6 , 34.5 , 31.6 , 31.3 , 30.0 , 29.1 , 29.1 , 26.7 , 26.5 , 25.9 , 25.3 , 24.3 , 23.5 , 22.7 , 22.2 , 21.9 , 20.8 , 20.4 , 19.9 , 19.6 , 19.4 , 18.8 , 14.2 , 11.5 ppm . hrms ( esi ) m / z calcd for c37h46cl3no9na ( m + na ) 776.2130 , found 776.2149 . to the solution of weinreb amide 28 ( 2.505 g , 4.945 mmol ) in thf ( 50 ml ) was added lialh4 ( 234.6 mg , 6.182 mmol ) in one portion at 0 c . after being stirred at 0 c for 30 min , the reaction mixture was quenched with 0.2n aq khso4 ( 30 ml ) and extracted with et2o ( 50 ml 3 ) . the combined organic layer was washed with 1n aq hcl ( 30 ml 3 ) and brine ( 30 ml 3 ) , dried with anhydrous mgso4 , and evaporated in vacuo to give the crude aldehyde 29 , which was used in the next step without further purification . to the crude aldehyde 28 in toluene ( 30 ml ) was added ph3p = chco2et ( 3.101 g , 8.901 mmol ) at 0 c under ar . after being stirred at room temperature for 3 h , the reaction mixture was concentrated in vacuo and purified by column chromatography ( eluted by 1217% ethyl acetate in hexane ) to give product(s ) 30 as a colorless oil . yield 88% , 2 steps ; [ ]d + 6.0 ( c 0.20 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 ) : 7.447.42 ( m , 6h ) , 7.317.27 ( m , 6h ) , 7.247.20 ( m , 3h ) , 6.72 ( dd , j = 15.6 , 4.4 hz , 1h ) , 5.84 ( d , j = 15.6 hz , 1h ) , 4.80 ( d , j = 8.4 hz , 1h ) , 4.29 ( br , 1h ) , 4.17 ( q , j = 7.2 hz , 2h ) , 2.532.41 ( m , 2h ) , 1.45 ( s , 9h ) , 1.27 ( t , j = 7.2 hz , 3h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 169.9 , 154.7 , 146.6 , 144.3 , 129.5 , 128.0 , 126.8 , 121.5 , 79.7 , 67.0 , 60.4 , 50.5 , 36.2 , 28.5 , 14.2 ppm . hrms ( esi ) m / z calcd for c31h35no4sna ( ( m + na ) 540.2210 , found 540.2223 . to the solution of compound 30 ( 3.379 g , 6.532 mmol ) in 95% etoh ( 35 ml ) was added nabh4 ( 247.1 mg , 6.532 mmol ) at room temperature . the resulting mixture was stirred at room temperature for 30 h and then quenched with water ( 50 ml ) and extracted with et2o ( 50 ml 4 ) . the combined organic layer was dried with anhydrous mgso4 , evaporated in vacuo , and purified by column chromatography ( eluted with the mixture of ch2cl2/hexane / acetone from 50:148:2 50:98:2 50:48:2 ) to give product 31 ( 1.187 g , 35% ) as a thick colorless oil . h nmr ( 400 mhz , cdcl3 ) : 7.427.40 ( m , 6h ) , 7.307.27 ( m , 6h ) , 7.237.19 ( m , 3h ) , 4.49 ( d , j = 9.2 hz , 1h ) , 4.10 ( q , j = 7.2 hz , 2h ) , 3.64 ( br , 1h ) , 2.34 ( br m , 2h ) , 2.21 ( t , j = 7.6 hz , 2h ) , 1.781.59 ( m , 2h ) , 1.43 ( s , 9h ) , 1.23 ( t , j = 7.2 hz , 3h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 173.3 , 155.3 , 144.7 , 129.7 , 128.0 , 126.8 , 79.4 , 66.7 , 60.5 , 49.6 , 37.2 , 31.1 , 29.7 , 28.5 , 14.3 ppm . hrms ( esi ) m / z calcd for c31h37no4sna ( m + na ) 542.2336 , found 542.2331 . to the solution of 31 ( 1.045 g , 2.013 mmol ) in 95% ethanol ( 7.5 ml ) was added aq naoh ( 1 m , 4.0 ml ) at room temperature . after being stirred at the same temperature for 2 h , the reaction mixture was diluted with water ( 10 ml ) , acidified with 10.5 m aq hcl to ph 45 , and extracted with diethyl ether ( 20 ml 3 ) . the combined organic layer was dried over mgso4 , filtered , and concentrated in vacuo to give crude acid 32 , which was used in the next step without further purification . to the above crude acid 32 solution in dmso ( 15 ml ) was added k2co3 ( 556.5 mg , 4.027 mmol ) and allyl bromide ( 255.5 l , 3.020 mmol ) at room temperature . after being stirred at room temperature 5 h , the reaction was quenched with water ( 30 ml ) and extracted with ethyl acetate ( 50 ml 3 ) . the combined organic layer was washed with brine , filtered , and concentrated in vacuo , and the residue was purified by column chromatography on silica gel ( eluted by 10% ethyl acetate in hexane ) to give product 5 as a colorless oil . yield 0.75 g , 70% : [ ]d + 10.0 ( c 0.16 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 ) : 7.427.41 ( m , 6h ) , 7.317.27 ( m , 6h ) , 7.237.20 ( m , 3h ) , 5.955.85 ( m , 1h ) , 5.325.21(m , 2h ) , 4.55 ( d , j = 5.6 hz , 2h ) , 4.49 ( d , j = 8.8 hz , 1h ) , 3.65 ( br m , 1h ) , 2.34 ( br m , 2h ) , 2.26 ( t , j = 7.6 hz , 2h ) , 1.801.62 ( m , 2h ) , 1.44 ( s , 9h ) ppm . c nmr ( 100 mhz , cdcl3 ) : 172.9 , 155.3 , 144.7 , 132.2 , 129.6 , 128.1 , 126.8 , 118.3 , 79.4 , 66.7,65.3 , 49.5 , 37.2 , 31.0 , 29.7 , 28.5 ppm . hrms ( esi ) m / z calcd for c32h37no4sna ( ( m + na ) 554.2336 , found 554.2341 . to the solution of 5 ( 169.6 mg , 0.319 mmol ) in ch2cl2 ( 4 ml ) were added 2,6-lutidine ( 556 l , 4.790 mmol ) and trimethylsilyl trifluoro - methanesulfonate ( tmsotf ) ( 694 l , 3.832 mmol ) dropwise at room temperature under argon . after being stirred at the same temperature for 1.5 h , the reaction mixture was quenched with meoh ( 6 ml ) and water ( 10 ml ) at 0 c and extracted with ch2cl2 ( 15 ml 4 ) . the combined organic layer was washed with brine , dried over anhydrous mgso4 , and evaporated in vacuo to give the crude free amine of 5 , which was used in next step without further purification . to the crude free amine of 5 ( 1.3 equiv ) in ch2cl2 ( 4 ml ) were added corresponding acid 4 ( based in scheme 5 ) ( 0.245 mmol , 1 equiv ) , coupling reagent ( cr , 0.368 mmol , 1.5 equiv ) , and diea ( 140 l , 0.735 mmol , 3.0 equiv ) at room temperature or 0 c . after being stirred at room temperature for 2.524 h , the resulting reaction mixture was evaporated in vacuo and purified by column chromatography , eluting with ethyl acetate in hexane to give product 3a , c , d or 34 as a colorless oil . from 4a , 5a ; cr bep added at 0 c ; reaction time 2.5 h ; yield 245.6 mg , 87% ) . from 4b , 5a ; cr hatu added at rt , reaction time 3.0 h ; yield 193.8 mg , 82% ; [ ]d 31.2 ( c 0.24 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.74 ( d , j = 7.6 hz , 2h ) , 7.637.54 ( m , 2h ) , 7.417.36 ( m , 8h ) , 7.307.15 ( m , 11h ) , 6.69 ( d , j = 8.8 hz , 0.84h ) , 6.06 ( d , j = 8.4 hz , 0.16h ) , 5.925.77 ( m , 1h ) , 5.305.15 ( m , 2h ) , 4.89 ( d , j = 11.2 hz , 0.84h ) , 4.79 ( d , j = 10.4 hz , 0.16h ) , 4.534.44 ( m , 2h ) , 4.394.29 ( m , 2h ) , 4.234.16 ( m , 1h ) , 4.01(br m , 1h ) , 3.953.85 ( m , 1h ) , 3.79 ( br , 1h ) , 3.663.61 ( m , 1h ) , 3.583.44 ( m , 1h ) , 2.402.15 ( m , 6h ) , 2.112.01 ( m , 3h ) , 1.981.88 ( m , 2h ) , 1.851.75 ( m , 2h ) , 1.711.60 ( m , 3h ) , 1.551.47 ( m , 1h ) , 1.401.30 ( m , 1h ) , 1.040.97 ( m , 1h ) , 0.96 ( d , j = 6.8 hz , 2.52h ) , 0.88 ( s , 9h ) , 0.77 ( d , j = 6.8 hz , 0.48h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 172.7 , 172.3 , 172.0 , 155.2 , 144.8 , 144.7 , 144.0,143.9 , 141.4 , 132.3 , 129.7 , 128.1 , 128.0 , 127.8 , 127.3 , 127.2 , 126.9 , 126.8 , 125.2 , 125.2 , 120.1 , 120.1 , 118.3 , 78.7 , 67.7 , 66.6 , 66.3 , 65.2 , 59.6 , 47.9 , 47.3 , 46.6 , 44.3 , 42.9 , 42.2 , 41.4 , 38.0 , 37.1 , 36.8 , 34.9 , 34.6 , 31.7 , 31.3 , 31.0 , 30.0 , 29.8 , 29.3 , 26.1 , 25.2 , 24.5 , 23.5 , 22.8 , 20.7 , 14.3 ppm . hrms ( esi ) m / z calcd for c59h68n2o8sna ( m + na ) 987.4589 , found 987.4628 . from 4c , 5a ; cr pyaop added at rt , reaction time 24 h ; yield 254.3 mg , 89% ; [ ]d 48.5 ( c 0.20 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.787.75 ( m , 2h ) , 7.67 ( d , j = 7.6 hz , 0.6h ) , 7.62 ( d , j = 7.2 hz , 1.4h ) , 7.417.38 ( m , 8h ) , 7.347.27 ( m , 8h ) , 7.227.20 ( m , 3h ) , 6.02 ( d , j = 8.0 hz , 0.4h ) , 5.96 ( d , j = 8.4 hz , 0.6 h ) , 5.925.80 ( m , 1h ) , 5.305.18 ( m , 2h ) , 5.13 ( d , j = 10.0 hz , 0.4h ) , 5.04 ( d , j = 10.0 hz , 0.6h ) , 4.95 ( d , j = 12.0 hz , 0.6h ) , 4.83 ( d , j = 12.0 hz , 0.4h ) , 4.794.75 ( m , 1h ) , 4.70 ( d , j = 12.0 hz , 0.4h ) , 4.66 ( d , j = 12.0 hz , 0.6h ) , 4.524.34 ( m , 4.4h ) , 4.284.23 ( m , 1h ) , 4.204.16 ( m , 0.6h ) , 3.993.87 ( m , 1h ) , 3.673.45 ( m , 2h ) , 2.362.27 ( m , 2.6h ) , 2.242.16 ( m , 2.4h ) , 2.12 ( br m , 1h ) , 2.011.90 ( m , 2h ) , 1.851.74 ( m , 3h ) , 1.531.44 ( m , 2h ) , 1.371.27 ( m , 1h ) , 1.191.16 ( m , 6h ) , 1.061.00 ( m , 1h ) , 0.97 ( d , j = 6.4 hz , 1.8h ) , 0.88 ( s , 5.4h ) , 0.85 ( s , 3.6h ) , 0.69 ( d , j = 6.4 hz , 1.2h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 174.2 , 172.7 , 172.6 , 172.2 , 154.6 , 154.3 , 154.2 , 154.1 , 144.5 , 144.5 , 144.3 , 144.1 , 143.9 , 143.7 , 141.3 , 141.2 , 132.1 , 132.0 , 129.5 , 128.0 , 127.7 , 127.1 , 127.0 , 126.8 , 125.4 , 125.3 , 125.1 , 125.1 , 120.0 , 119.9 , 118.3 , 94.8 , 94.8 , 81.6 , 81.4 , 79.6 , 79.4 , 79.3 , 76.7 , 67.8 , 67.4 , 66.5 , 65.2 , 59.8 , 59.3 , 48.3 , 47.2 , 47.0 , 46.8 , 46.3 , 38.1 , 37.8 , 36.8 , 36.3 , 36.3 , 34.8 , 34.7 , 34.5 , 31.6 , 31.3 , 30.8 , 30.0 , 26.8 , 26.5 , 25.8 , 25.8 , 25.3 , 24.4 , 23.4 , 23.1 , 22.7 , 20.4 , 20.3 , 20.1 , 14.2 ppm . hrms ( esi ) m / z calcd for c64h73cl3n2o10sna ( m + na ) 1189.3944 , found 1189.3964 . from 4a , 5b ; cr pyaop added at rt , reaction time 24 h ; yield 257.0 mg , 91% ; [ ]d 40.0 ( c 0.05 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.877.78 ( m , 2h ) , 7.717.67 ( dd , j = 8.4 , 8.0 hz , 2h),7.517.46 ( m , 8h ) , 7.407.28 ( m , 11h ) , 6.17 ( d , j = 8.4 hz , 0.6h ) , 6.025.92 ( m , 1h ) , 5.51 ( d , j = 8.4 hz , 0.4h ) , 5.405.30 ( m , 2h ) , 5.10 ( ddd , j = 7.6 , 7.4 , 7.2 hz , 1h ) , 4.974.80 ( m , 2.4h ) , 4.75 ( d , j = 12.0 hz , 0.6h ) , 4.624.30 ( m , 6h ) , 4.00 ( br m , 1h ) , 3.743.55 ( m , 2h ) , 2.602.57 ( m , 0.4h ) , 2.422.23 ( m , 6.6 h ) , 2.06 ( br m , 2h ) , 1.981.86 ( m , 1h ) , 1.821.70 ( m , 3h ) , 1.661.58 ( m , 2h ) , 1.551.30 ( m , 1h ) , 1.24 ( d , j = 6.8 hz , 1.8h ) , 1.20 ( d , j = 7.2 hz , 1.2 h ) , 1.07 ( d , j = 6.0 hz , 1.8h ) , 0.98 ( s , 3.6h ) , 0.96 ( s , 5.4h ) , 0.88 ( d , j = 6.4 hz , 1.2h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 172.8 , 172.8 , 172.6 , 172.5 , 172.4 , 172.3 , 154.9 , 154.5 , 153.7 , 153.7 , 144.6 , 144.5 , 144.2 , 144.0 , 143.8 , 141.4 , 141.2 , 132.2 , 129.8 , 129.6 , 128.1 , 128.0 , 127.8 , 127.3 , 127.2 , 127.1 , 126.9 , 126.9 , 125.7 , 125.5 , 125.2 , 120.0 , 118.6 , 118.5 , 79.5 , 79.2 , 79.0 , 78.7 , 76.7 , 68.0 , 67.5 , 66.8 , 66.7 , 65.4 , 65.4 , 59.8 , 59.5 , 48.1 , 47.3 , 47.3 , 47.1 , 46.4 , 45.7 , 45.4 , 38.3 , 37.6 , 37.1 , 36.6 , 36.5 , 35.1 , 34.8 , 34.8 , 31.7 , 31.4 , 29.9 , 30.1 , 29.8 , 29.2 , 29.2 , 26.2 , 26.1 , 26.0 , 25.9 , 25.4 , 24.4 , 23.5 , 22.8 , 20.6 , 20.2 , 14.3 , 14.0 , 13.8 ppm . hrms ( esi ) m / z calcd for c63h71cl3n2o10sna ( m + na ) 1175.3787 , found 1175.3805 . to the solution of compound 35 ( 166.3 mg , 0.172 mmol ) and pyridine ( 140 l , 1.742 mmol ) in ch2cl2 ( 5.0 ml ) were added 2,2,2-trichloroethoxylcarbonyl chloride ( troc - cl ) ( 182.6 mg/118.7 l , 0.862 mmol ) and 4-dimethylaminopyridine ( dmap)(0.36 mg , 2.93 mol ) at 0 c . after being stirred at the same temperature for 1 h the water layer was extracted with ethyl acetate ( 20 ml 4 ) . the combined organic layer was washed with 5% nahco3 ( 20 ml 2 ) and brine ( 20 ml ) , dried with mgso4 , and concentrated in vacuo . the residue was purified by chromatography column on silica gel ( 2030% ethyl acetate in hexane ) to give 3b ( 156.5 mg , 80% ) ; [ ]d 45.0 ( c 0.111 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.777.74 ( m , 2h ) , 7.67 ( d , j = 7.6 hz , 0.4h),7.60 ( d , j = 7.6 hz , 1.6h ) , 7.417.37 ( m , 8h ) , 7.337.17 ( m , 11h ) , 6.13 ( d , j = 8.4 hz , 0.6h ) , 5.925.80 ( m , 1h ) , 5.62 ( d , j = 8.4 hz , 0.4h ) , 5.305.18 ( m , 2.4h ) , 4.854.80 ( m , 1.2h ) , 4.77 ( d , j = 12.0 hz , 0.4h ) , 4.61 ( d , j = 12.0 hz , 0.4h ) , 4.54 ( d , j = 12.0 hz , 0.6h ) , 4.534.45 ( m , 3h ) , 4.404.30 ( m , 1.4h ) , 4.264.18 ( m , 1.6h ) , 3.933.82 ( m , 1h ) , 3.663.60 ( m , 1h ) , 3.583.45 ( m , 1h ) , 2.542.56 ( m , 4h ) , 2.222.10 ( m , 3h ) , 2.001.85 ( m , 3h ) , 1.751.60 ( m , 2h ) , 1.551.24 ( m , 3h ) , 0.99 ( d , j = 6.4 hz , 1.8h ) , 0.88 ( s , 3.6h ) , 0.87 ( s , 5.4h ) , 0.80 ( d , j = 6.4 hz , 1.2 h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 172.8 , 172.7 , 172.7 , 172.3 , 168.7 , 168.3 , 154.9 , 154.4 , 153.6 , 153.5 , 144.6 , 144.6 , 144.2 , 144.1 , 143.9 , 143.8 , 141.3 , 141.3 , 132.2 , 132.1 , 129.6 , 128.0 , 128.0 , 127.8 , 127.8 , 127.1 , 126.9 , 126.8 , 125.5 , 125.4 , 125.2 , 120.0 , 120.0 , 118.4 , 118.3 , 94.8 , 94.8 , 79.5 , 79.1 , 76.7 , 76.6 , 75.3 , 74.8 , 67.8 , 67.5 , 65.3 , 65.2 , 59.8 , 59.5 , 48.4 , 47.3 , 47.0 , 46.5 , 41.6 , 41.0 , 39.8 , 39.4 , 38.1 , 37.7 , 36.4 , 36.3 , 35.0 , 34.7 , 31.7 , 31.3 , 29.8 , 29.1 , 25.9 , 25.9 , 25.4 , 24.4 , 23.4 , 22.7 , 20.8 , 20.7 , 14.2 ppm . hrms ( esi ) m / z calcd for c62h69cl3n2o10sna ( m + na ) 1161.3631 , found 1161.3634 . to the solution of triphenylphosphine oxide ( 223.3 mg , 0.802 mmol ) in ch2cl2 ( 1 ml ) was added dropwise trifluoromethanesulfonic anhydride ( tf2o ) ( 68 l , 0.401 mmol ) at 0 c under argon . after being stirred at the same temperature for 10 min , compound 3 ( 0.100 mmol ) in ch2cl2 ( 0.5 ml ) the reaction mixture was stirred at the same temperature for 30 min for 3a/3b and 3 h for 3d . this reaction was monitored by mass spectrometry and was quenched with saturated nahco3 ( 6 ml ) at 0 c when starting material disappeared . the aqueous layer was extracted with ethyl acetate ( 10 ml 4 ) , washed with brine ( 10 ml ) , dried with mgso4 , filtered , and concentrated in vacuo to give troc protected thiazoline intermediate 33a , b , or d. the residue was used in the next step immediately without further purification . the above residue 33 was dissolved in thf ( 4 ml ) , and then aqueous nh4oac ( 1 m , 1.0 ml ) and zinc powder ( freshly activated with 1 m aqueous hcl ) ( 100 mg ) were added at room temperature . after being stirred at the same temperature for 30 min , ethyl acetate ( 5 ml ) and brine ( 5 ml ) and the aqueous layer was extracted with ethyl acetate ( 5 ml 4 ) . the combined organic layer was dried with mgso4 , filtered , concentrated in vacuo , and purified by column chromatography on silica gel ( eluted by ethyl acetate / hexane 1:3 , v / v ) to give thiazoline ring product 34a , b , or d as a colorless oil . yield 53.2 mg , 76% ; [ ]d 78.0 ( c 0.10 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.74 ( d , j = 7.6 hz , 2h ) , 7.63 ( dd , j = 5.6 , 6.0 hz , 1.6 h ) , 7.56 ( d , j = 7.6 hz , 0.4h ) , 7.37 ( dd , j = 7.2 , 7.2 hz , 2h ) , 7.327.27 ( m , 2h ) , 5.935.83 ( m , 1h ) , 5.305.19 ( m , 2h ) , 4.90 ( d , j = 10.8 hz , 0.8h ) , 4.83 ( d , j = 9.6 hz , 0.2h ) , 4.584.51 ( m , 2h ) , 4.464.34 ( m , 3.8h ) , 4.284.18 ( m , 1.2h ) , 3.97 ( br m , 1h ) , 3.683.61 ( m , 2h ) , 3.573.47 ( m , 1h ) , 3.33 ( dd , j = 10.4 , 8.8 hz , 0.2h ) , 3.24 ( dd , j = 10.4 , 8.8 hz , 0.8h ) , 2.88 ( dd , j = 10.0 , 8.4 hz , 0.2h ) , 2.81 ( dd , j = 10.4 , 8.4 hz , 0.8h ) , 2.552.48 ( m , 4h ) , 2.292.19 ( m , 1h ) , 2.081.80 ( m , 5h ) , 1.751.58 ( m , 2h ) , 1.481.25 ( m , 2h ) , 1.040.97 ( m , 1h ) , 0.96 ( d , j = 6.4 hz , 2.4h ) , 0.87 ( s , 9h ) , 0.79 ( d , j = 6.4 hz , 0.6h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 172.9 , 172.9 , 172.8 , 172.3 , 169.2 , 169.1 , 154.9 , 154.3 , 144.3 , 144.1 , 144.0 , 143.8 , 141.3 , 141.3 , 141.2 , 132.2 , 132.2 , 127.6 , 127.6 , 127.1 , 127.0 , 127.0 , 127.0 , 125.4 , 125.3 , 125.3 , 125.2 , 79.5 , 78.5 , 76.0 , 75.8 , 67.8 , 67.7 , 67.2 , 66.7 , 65.2 , 65.1 , 59.5 , 47.2 , 47.1 , 47.0 , 46.5 , 42.9 , 42.2 , 42.0 , 41.9 , 38.0 , 37.9 , 37.4 , 34.8 , 34.7 , 34.6 , 31.6 , 31.5 , 31.4 , 31.2 , 30.4 , 29.9 , 26.0 , 25.9 , 25.7 , 25.3 , 25.1 , 24.5 , 23.3 , 22.7 , 20.6 , 20.5 , 14.2 ppm . hrms ( esi ) m / z calcd for c40h52n2o7sna ( m + na ) 727.3387 , found 727.3399 . yield 57.0 mg , 79% ; [ ]d 22.5 ( c 0.12 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.76 ( d , j = 7.2 hz , 2h ) , 7.63 ( dd , j = 6.6 , 6.4 hz , 1.7 h ) , 7.57 ( d , j = 7.2 hz , 0.3h ) , 7.39 ( dd , j = 7.2 , 7.2 hz , 2h ) , 7.30 ( dd , j = 7.4 , 7.2 hz , 2h ) , 5.945.85 ( m , 1h ) , 5.315.20 ( m , 2h ) , 4.88 ( d , j = 10.0 hz , 0.7h ) , 4.81 ( d , j = 8.8 hz , 0.3h ) , 4.594.51 ( m , 2.3h ) , 4.454.33 ( m , 3h ) , 4.314.18 ( m , 1.7h ) , 3.823.72 ( m , 0.7h ) , 3.683.62 ( m , 1.3h ) , 3.583.49 ( m , 1.3h ) , 3.353.24 ( m , 1h ) , 2.902.85 ( m , 0.3h ) , 2.81 ( dd , j = 10.8 , 7.6 hz , 0.7h ) , 2.722.65 ( m , 0.7h ) , 2.612.44 ( m , 2.3h ) , 2.342.18 ( m , 1.7h ) , 2.101.89 ( m , 4.4h ) , 1.82 ( m , 0.7h ) , 1.701.57 ( m , 2h ) , 1.371.29 ( m , 1h ) , 1.19 ( d , j = 7.0 hz , 0.9h ) , 1.17 ( d , j = 7.0 hz , 2.1h ) , 1.061.00 ( m , 1h ) , 0.96 ( d , j = 6.4 hz , 2.1h ) , 0.87 ( s , 9h ) , 0.79 ( d , j = 6.4 hz , 0.9h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 174.2 , 173.1 , 173.0 , 172.8 , 172.5 , 155.0 , 154.5 , 144.4 , 144.3 , 144.1 , 143.9 , 141.4 , 141.4 , 141.3 , 132.3 , 132.3 , 128.0 , 127.7 , 127.4 , 127.2 , 127.1 , 125.6 , 125.4 , 125.3 , 125.3 , 121.0 , 120.0 , 118.4 , 118.3 , 79.6 , 78.6 , 77.4 , 76.2 , 75.7 , 71.5 , 71.5 , 70.7 , 67.9 , 67.8 , 65.3 , 65.3 , 59.7 , 59.7 , 47.3 , 47.2 , 47.1 , 46.6 , 46.0 , 45.3 , 40.4 , 39.3 , 38.2 , 37.8 , 37.4 , 37.3 , 34.8 , 34.7 , 31.7 , 31.5 , 31.3 , 30.3 , 30.2 , 30.1 , 29.8 , 26.1 , 26.1 , 25.9 , 25.5 , 25.3 , 24.6 , 23.4 , 20.7 , 20.6 , 16.3 , 15.8 ppm . hrms ( esi ) m / z calcd for c41h54n2o7sna ( m + na ) 741.3544 , found 741.3567 . to the solution of 3c ( 97.8 mg , 0.084 mmol ) in 1,2-dichloroethane ( 5 ml ) was added ticl4 ( 1 m in ch2cl2 , 0.294 ml , 0.293 mmol , 3.5 equiv ) at room temperature . the resulting solution was heated to 60 c and stirred at this temperature for 2.5 h. this reaction was monitored by mass spectrometry . when the starting material was consumed completely , the reaction was cooled to 0 c and quenched with saturated aq nahco3 ( 7 ml ) , stirred at room temperature for another 10 min , extracted with ethyl acetate ( 10 ml 4 ) , dried over anhydrous mgso4 , filtered , and evaporated in vacuo to give crude intermediate 33c , which was used in the next step without further purification . the above crude 33c was dissolved in thf ( 4 ml ) , and then aqueous nh4oac ( 1 m , 1.0 ml ) and zinc powder ( freshly activated with 1 m aqueous hcl ) ( 80 mg ) were added at room temperature . after being stirred at the same temperature for 30 min , to the reaction the aqueous layer was extracted with ethyl acetate ( 7 ml 4 ) . the combined organic layer was dried with mgso4 , filtered , concentrated in vacuo , and purified by column chromatography on silica gel ( eluted by ethyl acetate / hexane 1:3 , v / v ) to give thiazoline ring product 34c ( 101.3 mg , 55% ) as a colorless oil ; [ ]d 80.8 ( c 0.12 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.767.74 ( m , 2h ) , 7.667.61 ( m , 1.7h ) , 7.57 ( d , j = 7.2 hz , 0.3h ) , 7.39 ( dd , j = 7.4 , 7.2 hz , 2h ) , 7.317.28 ( m , 2h ) , 5.945.84 ( m , 1h ) , 5.315.19 ( m , 2h ) , 4.88 ( dd , j = 11.6 , 1.6 hz , 0.7h ) , 4.81 ( dd , j = 11.6 , 1.6 hz , 0.3h ) , 4.584.54 ( m , 2h ) , 4.524.38 ( m , 3h ) , 4.354.19 ( m , 2h ) , 3.663.59 ( m , 2h ) , 3.583.48 ( m , 2h ) , 3.303.23 ( m , 1h ) , 2.862.81 ( m , 1h ) , 2.592.46 ( m , 2h ) , 2.362.19 ( m , 1h ) , 2.121.91 ( m , 5h ) , 1.78 ( br m , 1h ) , 1.671.56 ( m , 2h ) , 1.371.27 ( m , 2h ) , 1.22 ( s , 0.9h ) , 1.191.18 ( m , 5.1h ) , 0.96 ( d , j = 6.4 hz , 2.1h ) , 0.87 ( s , 9h ) , 0.79 ( d , j = 6.8 hz , 0.9h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 178.8 , 178.3 , 173.1 , 173.0 , 172.9 , 172.5 , 154.9 , 154.4 , 144.4 , 144.3 , 144.0 , 143.9 , 141.4 , 141.3 , 141.2 , 132.3 , 127.7 , 127.7 , 127.1 , 127.1 , 125.5 , 125.4 , 125.3 , 125.2 , 120.0 , 118.3 , 118.3 , 79.4 , 78.6 , 76.0 , 75.8 , 75.0 , 74.9 , 67.9 , 67.7 , 65.2 , 65.2 , 59.7 , 59.7 , 47.3 , 47.2 , 47.0 , 46.5 , 45.9 , 45.6 , 38.0 , 37.8 , 37.0 , 37.0 , 36.9 , 36.6 , 34.8 , 34.6 , 31.6 , 31.4 , 31.3 , 30.3 , 30.2 , 30.0 , 29.8 , 26.1 , 25.8 , 25.3 , 24.5 , 24.3 , 23.7 , 23.4 , 23.1 , 20.6 , 20.5 ppm . hrms ( esi ) m / z calcd for c42h56n2o7sna ( m + na ) 755.3700 , found 755.3718 . to a solution of 34 ( 0.054 mmol ) in thf ( 1.5 ml ) were added pd(pph3)4 ( 7.1 mg , 0.005 mmol ) and n - methylaniline ( 0.015 ml , 0.136 mmol ) at room temperature under argon . this reaction was protected with aluminum foil . after being stirred at the same temperature for 1 h , the reaction mixture was concentrated in vacuo and purified by preparative tlc ( 20 cm 20 cm plate ) to give acid 2 . yield 31.8 mg , 89% ; [ ]d 90.0 ( c 0.04 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.74 ( d , j = 7.6 hz , 2h ) , 7.63(d , j = 7.2 hz , 1.8h ) , 7.57 ( d , j = 7.6 hz , 0.2h ) , 7.38 ( dd , j = 7.6 , 7.6 hz , 2h ) , 7.327.28 ( m , 2h ) , 6.22 ( br , 1h ) , 4.90 ( d , j = 10.0 hz , 0.8h ) , 4.84 ( d , j = 9.2 hz , 0.2h ) , 4.534.34 ( m , 3.8h ) , 4.294.17 ( m , 1.2h ) , 4.003.92 ( m , 1h ) , 3.683.61 ( m , 1h ) , 3.573.45 ( m , 1h ) , 3.35 ( dd , j = 10.8 , 8.8 hz , 0.2h ) , 3.23 ( dd , j = 10.8 , 8.4 hz , 0.8h ) , 2.91 ( dd , j = 10.8 , 8.4 hz , 0.2h ) , 2.80 ( dd , j = 10.8 , 8.4 hz , 0.8h ) , 2.602.43 ( m , 4h ) , 2.352.17 ( m , 1h ) , 2.081.82 ( m , 5h ) , 1.741.59 ( m , 2h ) , 1.481.25 ( m , 2h ) , 1.030.96 ( m , 1h ) , 0.96 ( d , j = 6.4 hz , 2.4h ) , 0.87 ( s , 9h ) , 0.77 ( d , j = 6.4 hz , 0.6h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 176.7 , 176.5 , 173.1 , 172.5 , 171.8 , 170.5 , 155.1 , 154.5 , 144.3 , 144.1 , 144.0 , 143.8 , 141.4 , 141.4 , 141.3 , 141.3 , 127.7 , 127.7 , 127.2 , 127.1 , 127.1 , 125.5 , 125.4 , 125.3 , 120.0 , 79.8 , 75.7 , 75.3 , 67.9 , 67.8 , 67.4 , 67.0 , 59.6 , 47.2 , 47.2 , 47.0 , 46.6 , 42.7 , 42.3 , 42.1 , 41.9 , 38.2 , 38.1 , 38.0 , 37.4 , 36.8 , 34.8 , 34.7 , 32.0 , 31.7 , 31.3 , 30.1 , 30.0 , 30.0 , 26.1 , 26.0 , 25.7 , 25.1 , 24.6 , 23.4 , 20.7 , 20.5 ppm . hrms ( esi ) m / z calcd for c37h48n2o7sna ( m + na ) 687.3074 , found 687.3070 . yield 31.2 mg , 84% ; [ ]d 57.7 ( c 0.052 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.76 ( d , j = 7.6 hz , 2h ) , 7.657.58 ( m , 2h ) , 7.39 ( dd , j = 7.4 , 7.2 hz , 2h ) , 7.327.28 ( m , 2h ) , 4.88 ( d , j = 12.0 , 0.7h ) , 4.80 ( d , j = 12.0 hz , 0.3h ) , 4.51 ( dd , j = 8.8 , 2.8 hz , 0.3h ) , 4.46 ( d , j = 7.2 hz , 0.3h ) , 4.43 ( d , j = 7.2 hz , 0.7h ) , 4.414.32 ( m , 2h ) , 4.304.25 ( m , 1.7h ) , 3.653.56 ( m , 2h ) , 3.533.45 ( m , 1h ) , 3.30 ( dd , j = 11.0 , 8.4 hz , 0.3h ) , 3.19 ( dd , j = 11.0 , 8.4 hz , 0.7h ) , 2.88 ( dd , j = 10.8 , 10.8 hz , 1h ) , 2.702.53 ( m , 2h ) , 2.372.14 ( m , 1h ) , 2.081.90 ( m , 5h ) , 1.79 ( br m , 1h ) , 1.681.60 ( m , 2h ) , 1.351.26 ( m , 2h ) , 1.26 ( s , 0.9h ) , 1.19 ( s , 2.1h ) , 1.18 ( s , 3h ) , 0.95 ( d , j = 6.6 hz , 2.1h ) , 0.87 ( s , 9h ) , 0.74 ( d , j = 6.6 hz , 0.9h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 182.4 , 180.5 , 175.7 , 175.5 , 173.2 , 172.7 , 155.1 , 154.7 , 144.5 , 144.2 , 144.1 , 143.8 , 141.5 , 141.4 , 141.3 , 127.8 , 127.8 , 127.2 , 127.1 , 127.1 , 125.5 , 125.4 , 125.4 , 125.3 , 120.0 , 79.7 , 78.6 , 76.0 , 75.8 , 75.4 , 75.1 , 68.1 , 67.9 , 59.7 , 59.6 , 47.3 , 47.2 , 47.1 , 46.6 , 46.5 , 45.8 , 38.0 , 37.8 , 37.5 , 37.4 , 37.1 , 36.3 , 34.8 , 34.6 , 33.4 , 32.7 , 32.7 , 31.7 , 31.3 , 30.2 , 30.0 , 29.8 , 29.7 26.1 , 25.6 , 25.1 , 24.9 , 24.7 , 23.8 , 23.7 , 23.5 , 22.8 , 20.7 , 20.4 , 14.3 ppm . hrms ( esi ) m / z calcd for c39h52n2o7sna ( m + na ) 715.3387 , found 715.3397 . yield 33.0 mg , 90% ; [ ]d 60.0 ( c 0.04 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.75 ( d , j = 7.6 hz , 2h ) , 7.677.57 ( m , 2h ) , 7.39 ( dd , j = 7.4 , 7.2 hz , 2h ) , 7.30 ( dd , j = 7.6 , 7.4 hz , 2h ) , 5.90 ( br , 1h ) , 4.91(dd , j = 12.8 , 12.4 hz , 0.7h ) , 4.864.82 ( m , 0.3h ) , 4.544.50 ( m , 0.3h ) , 4.454.40 ( m , 1h ) , 4.384.33 ( m , 1.7h ) , 4.304.27 ( m , 1.3h ) , 4.254.16 ( m , 0.7h ) , 3.823.80 ( m , 0.7h ) , 3.753.70 ( m , 0.3h ) , 3.64 ( br m , 1h ) , 3.513.49 ( m , 1h ) , 3.363.30 ( m , 0.3h ) , 3.263.15 ( m , 0.7h ) , 2.912.85 ( m , 0.3h ) , 2.842.72 ( m , 0.7h ) , 2.682.50 ( m , 3h ) , 2.352.21 ( m , 1h ) , 2.212.15 ( m , 0.3h ) , 2.051.89 ( m , 5h ) , 1.841.79 ( m , 0.7h ) , 1.751.70 ( m , 1h ) , 1.661.60 ( m , 1h ) , 1.541.45 ( m , 0.7h ) , 1.451.40 ( m , 0.3h ) , 1.191.16 ( m , 3h ) , 1.031.00 ( m , 1h ) , 0.96 ( d , j = 6.0 hz , 2.1h ) , 0.88 ( s , 9h ) , 0.74 ( d , j = 6.0 hz , 0.9h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 177.8 , 177.6 , 175.9 , 173.4 , 173.4 , 173.3 , 172.7 , 155.1 , 155.1 , 154.6 , 144.4 , 144.4 , 144.2 , 144.2 , 144.2 , 144.0 , 143.9 , 141.4 , 141.4 , 141.4 , 141.3 , 141.3 , 128.0 , 128.0 , 127.8 , 127.8 , 127.5 , 127.2 , 127.1 , 125.5 , 125.4 , 125.4 , 125.3 , 121.0 , 121.0 , 120.0 , 120.0 , 80.0 , 80.0 , 79.2 , 78.7 , 77.4 , 76.2 , 76.0 , 75.6 , 75.5 , 71.8 , 70.8 , 70.4 , 68.0 , 67.9 , 67.9 , 67.7 , 67.5 , 59.7 , 59.6 , 47.3 , 47.3 , 47.1 , 46.6 , 46.0 , 45.9 , 45.4 , 40.7 , 39.7 , 39.4 , 39.2 , 38.1 , 38.0 , 37.8 , 37.6 , 37.0 , 34.8 , 34.7 , 32.9 , 32.9 , 32.3 , 31.7 , 31.3 , 31.1 , 30.2 , 30.0 , 30.0 , 29.8 , 29.4 , 29.2 , 28.7 , 26.1 , 26.0 , 25.8 , 25.4 , 25.2 , 25.1 , 24.6 , 23.4 , 22.8 , 20.8 , 20.8 , 20.6 , 20.5 , 16.6 , 16.0 , 14.3 , 14.2 ppm . hrms ( esi ) m / z calcd for c38h50n2o7sna ( m + na ) 701.3231 , found 701.3253 . to a solution of fmoc protected tripeptide 6(2a2c ) ( 28.1 mg , 0.042 mmol ) in mecn ( 1.2 ml ) was added diethylamine ( 0.6 ml ) at room temperature . after being stirred at the same temperature for 30 min , the reaction mixture was evaporated in vacuo , then azeotroped with toluene and ch2cl2 two times , respectively , and dried under reduced pressure for 1 h to give the free amine tripeptide , which was used in the next coupling reaction without further purification . the above crude free amine tripeptide was dissolved in ch2cl2 ( thf for 2b ) ( 2 ml ) . to this solution were added acid 2 ( 0.028 mmol ) , corresponding coupling reagent ( 0.056 mmol ) ( pyaop for 2a , c , d and depbt for 2b ) , and diea ( 0.014 ml , 0.083 mmol ) at room temperature . after being stirred at the same temperature for 1524 h , the reaction mixture was concentrated in vacuo and purified by preparative tlc plate ( developed by acetone / hexane ( 2:3 , v / v ) ) to give the precursor 36a d as a colorless oil . yield 22.0 mg , 72% ; [ ]d 99.3 ( c 0.14 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.777.74 ( m , 2h ) , 7.647.62 ( m , 1.7h ) , 7.56 ( d , j = 7.6 hz , 0.3h ) , 7.39 ( dd , j = 7.6 , 7.4 hz , 2h ) , 7.327.26 ( m , 2h ) , 7.09 ( d , j = 8.0 hz , 2h ) , 6.76 ( d , j = 8.0 hz , 2h ) , 6.63 ( d , j = 8.0 hz , 0.3h ) , 6.49 ( br m , 0.3h ) , 6.346.27 ( m , 1h ) , 6.206.12 ( m , 0.3h ) , 5.925.83 ( m , 0.7h ) , 5.545.47 ( m , 0.3h ) , 5.425.35 ( m , 0.7h ) , 5.315.17 ( m , 2.7h ) , 5.115.06 ( m , 0.3h ) , 4.944.85 ( m , 1.7h ) , 4.624.50 ( m , 2h ) , 4.474.35 ( m , 2.3h ) , 4.27 ( t , j = 7.2 hz , 1h ) , 4.204.16 ( m , 1.4h ) , 3.74 ( s , 3h ) , 3.723.64 ( m , 1.3h ) , 3.603.54 ( m , 1h ) , 3.263.07 ( m , 1h ) , 3.022.90 ( m , 4h ) , 2.832.77 ( m , 2h ) , 2.772.60 ( m , 4h ) , 2.502.46 ( m , 0.7h ) , 2.392.19 ( m , 4h ) , 2.181.90 ( m , 6.3h ) , 1.871.78 ( m , 1h ) , 1.73 ( br m , 1h ) , 1.61 ( br m , 0.7h ) , 1.551.49 ( m , 1h ) , 1.421.38 ( m , 1h ) , 1.271.21 ( m , 3h ) , 1.000.84 ( m , 19.1h ) , 0.68 ( d , j = 6.4 hz , 0.9h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 172.7 , 172.1 , 172.0 , 171.9 , 171.6 , 171.6 , 170.8 , 158.7 , 154.8 , 154.4 , 144.3 , 144.2 , 144.1 , 143.9 , 141.4 , 141.3 , 131.8 , 131.4 , 130.5 , 130.4 , 128.2 , 128.2 , 127.8 , 127.8 , 127.2 , 127.1 , 127.1 , 126.4 , 125.5 , 125.4 , 125.3 , 120.1 , 118.8 , 114.0 , 79.6 , 79.1 , 77.4 , 75.9 , 75.7 , 67.9 , 67.7 , 66.1 , 65.5 , 60.6 , 59.6 , 59.4 , 55.3 , 50.4 , 49.8 , 49.2 , 47.3 , 47.1 , 46.6 , 38.7 , 38.2 , 37.9 , 37.2 , 37.1 , 36.8 , 34.8 , 34.5 , 33.7 , 33.4 , 32.1 , 31.7 , 31.5 , 31.1 , 31.0 , 30.7 , 30.2 , 29.8 , 29.6 , 29.5 , 28.8 , 26.1 , 25.1 , 24.6 , 23.5 , 22.8 , 21.1 , 20.7 , 16.2 , 15.9 , 14.5 , 14.4 , 14.3 , 11.8 , 10.7 ppm . hrms ( esi ) m / z calcd for c61h83n5o11sna ( m + na ) 1116.5702 , found 1116.5742 . yield 28.3 mg , 90% ; [ ]d 130.0 ( c 0.10 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.767.73 ( m , 2h ) , 7.657.59 ( m , 2h ) , 7.407.36 ( m , 2h ) , 7.317.26 ( m , 2h ) , 7.107.08 ( m , 2h ) , 6.85 ( d , j = 8.4 hz , 0.3h ) , 6.776.74 ( m , 2h ) , 6.65 ( d , j = 8.0 hz , 0.7h ) , 5.925.82 ( m , 1h ) , 5.37 ( dddd , j = 6.8 , 6.8 , 6.8 , 6.8 hz , 1h ) , 5.315.20 ( m , 2h ) , 5.205.14 ( m , 1h ) , 4.934.87 ( m , 1.7h ) , 4.81 ( d , j = 10.8 hz , 0.3h ) , 4.624.51 ( m , 3h ) , 4.464.41 ( m , 2h ) , 4.334.24 ( m , 3h ) , 3.74 ( s , 3h ) , 3.683.62 ( m , 1.3h ) , 3.603.48 ( m , 1.7h ) , 3.18 ( ddd , j = 18.8 , 10.8 , 8.8 hz , 1h ) , 3.032.95 ( m , 4h ) , 2.822.73 ( m , 2h ) , 2.68 ( s , 2.1h ) , 2.65 ( s , 0.9h ) , 2.432.17 ( m , 4h ) , 2.151.86 ( m , 5h ) , 1.851.75 ( m , 2h ) , 1.701.55 ( m , 2.3h ) , 1.501.45 ( m , 0.7h ) , 1.351.17 ( m , 13h ) , 0.970.91 ( m , 6h ) , 0.910.82 ( m , 14.1h ) , 0.76 ( d , j = 6.4 hz , 0.9h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 178.8 , 178.3 , 173.0 , 172.7 , 172.3 , 172.2 , 171.9 , 171.6 , 171.6 , 171.4 , 170.8 , 169.7 , 158.6 , 154.9 , 154.6 , 144.6 , 144.3 , 144.0 , 143.9 , 141.4 , 141.4 , 141.3 , 131.8 , 130.5 , 130.5 , 130.4 , 128.4 , 128.3 , 127.8 , 127.7 , 127.1 , 125.5 , 125.4 , 125.4 , 125.3 , 120.0 , 120.0 , 118.8 , 113.9 , 79.4 , 78.7 , 77.4 , 76.1 , 75.7 , 75.2 , 75.0 , 68.0 , 67.7 , 66.1 , 65.5 , 64.6 , 60.5 , 59.8 , 59.6 , 55.3 , 53.6 , 50.4 , 49.8 , 49.3 , 47.3 , 47.2 , 47.1 , 46.6 , 45.9 , 45.4 , 38.1 , 37.9 , 37.0 , 36.6 , 34.8 , 34.7 , 34.5 , 33.7 , 33.5 , 33.3 , 32.0 , 31.7 , 31.3 , 31.0 , 31.0 , 30.9 , 30.6 , 30.1 , 29.8 , 26.1 , 25.6 , 25.4 , 25.1 , 24.6 , 24.0 , 23.7 , 23.5 , 22.8 , 20.8 , 20.7 , 20.6 , 16.2 , 15.9 , 15.0 , 14.9 , 14.4 , 14.3 , 11.7 , 10.7 ppm . hrms ( esi ) m / z calcd for c63h87n5o11sna ( m + na ) 1144.6015 , found 1144.6041 . yield 22.0 mg , 71% ; [ ]d 66.0 ( c 0.05 , ch2cl2 ) . h nmr ( 400 mhz , cdcl3 , mixture of rotamers ) : 7.777.74 ( m , 2h ) , 7.657.60 ( m , 2h ) , 7.39 ( dd , j = 7.6 , 7.2 hz , 2h ) , 7.30 ( dd , j = 7.2 , 6.8 hz , 2h ) , 7.107.07 ( m , 2h ) , 6.786.75 ( m , 2h ) , 5.925.81 ( m , 1h ) , 5.395.35 ( m , 1h ) , 5.315.21 ( m , 2h ) , 5.185.11 ( m , 1h ) , 4.934.81 ( m , 2h ) , 4.624.53 ( m , 2h ) , 4.464.21 ( m , 5h ) , 3.813.69 ( m , 4h ) , 3.693.60 ( m , 1h ) , 3.583.49 ( m , 1h ) , 3.223.18 ( m , 1h ) , 3.032.88 ( m , 4h ) , 2.822.76 ( m , 3h ) , 2.712.57 ( m , 4h ) , 2.342.21 ( m , 4h ) , 2.111.79 ( m , 8h ) , 1.71 ( br m , 1h ) , 1.641.58 ( m , 1h ) , 1.341.18 ( m , 5h ) , 0.970.78 ( m , 21h ) ppm . c nmr ( 100 mhz , cdcl3 , mixture of rotamers ) : 175.1 , 174.6 , 173.2 , 172.9 , 172.7 , 172.6 , 172.3 , 172.2 , 172.0 , 172.0 , 171.9 , 171.9 , 171.8 , 171.6 , 171.5 , 171.4 , 170.8 , 169.7 , 158.7 , 158.6 , 155.1 , 155.0 , 154.5 , 144.5 , 144.4 , 144.3 , 144.2 , 144.1 , 144.0 , 143.9 , 141.4 , 141.4 , 141.3 , 131.8 , 130.5 , 128.5 , 128.3 , 128.0 , 127.8 , 127.1 , 125.5 , 125.4 , 125.4 , 125.3 , 120.0 , 118.8 , 114.0 , 113.9 , 79.7 , 78.8 , 77.4 , 76.2 , 75.9 , 75.6 , 71.8 , 70.9 , 70.3 , 67.9 , 67.8 , 67.6 , 66.1 , 65.5 , 60.5 , 59.8 , 59.7 , 59.7 , 55.3 , 50.4 , 49.9 , 47.3 , 47.3 , 47.2 , 47.1 , 46.6 , 45.8 , 45.2 , 45.0 , 40.8 , 39.8 , 39.6 , 38.1 , 37.8 , 37.7 , 37.3 , 37.3 , 34.8 , 34.7 , 33.5 , 33.4 , 32.1 , 31.7 , 31.4 , 31.0 , 31.0 , 30.7 , 30.6 , 30.1 , 30.1 , 29.8 , 26.1 , 25.7 , 25.4 , 25.2 , 25.1 , 24.6 , 23.4 , 22.8 , 20.7 , 20.6 , 16.8 , 16.4 , 15.9 , 14.4 , 14.4 , 14.3 , 13.9 , 11.8 , 11.6 , 10.7 ppm . hrms ( esi ) m / z calcd for c62h85n5o11sna ( m + na ) 1130.5859 , found 1130.5905 . to a solution of cyclic precursor 36 ( 11.6 mol ) in thf ( 1.0 ml ) were added pd(pph3)4 ( 2.7 mg , 2.32 mol ) and n - methylaniline ( 6.3 l , 58.0 mol ) at room temperature under argon . this reaction was protected with aluminum foil . after being stirred at the same temperature for 1 h , the reaction mixture was concentrated in vacuo and purified by preparative tlc plate ( developed with meoh / ch2cl2 1:9 , v / v ) to give the free acid cyclic precursor . to the solution of free acid cyclic precursor in mecn ( 1.5 ml ) was added n , n - diethylamine ( 0.75 ml ) . after being stirred at room temperature for 30 min , the reaction mixture was evaporated in vacuo , azeotroped with toluene ( three times ) and ch2cl2 ( two times ) , and then dried under reduced pressure for 1 h to give the unmasked precursor as a foam solid . then the unmasked precursor was dissolved in ch2cl2 ( dmf for 36b ) ( 20 ml ) . to this solution were added diea ( 20.0 l , 0.116 mmol ) and corresponding coupling reagent ( 34.8 mol ) at 0 c ( pyaop for 36a , c , d and depbt for 36b ) . after being stirred at 0 c for 30 min , the reaction was allowed to warm up to room temperature and stirred for additional 15 h. then the reaction was concentrated in vacuo and purified by semipreparative reversed - phase hplc ( phenomenex ultracarb , ods 250 mm 10 mm , 5 m , 3.0 ml / min , uv detection at 200/220 nm ) using an isocratic system of 80% aqueous mecn for 30 min , 80100% mecn for 3040 min , and 100% mecn for 4060 min to afford 1a d . yield 2.3 mg , 25% in 3 steps ; [ ]d 106.2 ( c 0.024 , ch2cl2 ) ; tr = 17.5 min . h nmr ( 600 mhz , cdcl3 , mixture of rotamers , major and minor ( 6/4 ) ) : 7.14 ( d , j = 8.4 hz , 1.2h ) , 7.10 ( d , j = 8.4 hz , 0.8h ) , 6.80 ( d , j = 8.4 hz , 0.8h ) , 6.79 ( d , j = 8.4 hz , 1.2h ) , 6.17 ( d , j = 9.0 hz , 0.4h ) , 5.78 ( d , j = 10.2 hz , 0.6h ) , 5.26 ( d , j = 11.4 hz , 0.6h ) , 5.205.16 ( m , 0.4h ) , 5.15 ( ddd , j = 15.0 , 10.2 , 4.8 hz , 0.6h ) , 4.96 ( dd , j = 12.6 , 2.4 hz , 0.6h ) , 4.89 ( d , j = 11.4 hz , 0.4h ) , 4.87 ( dd , j = 12.6 , 2.4 hz , 0.4h ) , 4.64 ( q , j = 6.6 hz , 0.4h ) , 4.60 ( d , j = 10.8 hz , 0.6h ) , 4.384.31 ( m , 1.2h ) , 4.244.19 ( m , 1h ) , 4.114.08 ( m , 0.4h ) , 4.05 ( br m , 0.4h ) , 4.003.95 ( m , 0.6h ) , 3.90 ( br m , 0.4h ) , 3.77 ( s , 1.8h ) , 3.77 ( s , 1.2h ) , 3.703.66 ( m , 0.6h ) , 3.653.61 ( m , 0.4h ) , 3.39 ( dd , j = 10.8 , 8.4 hz , 0.6h ) , 3.32 ( dd , j = 10.8 , 8.4 hz , 0.4h ) , 3.29 ( q , j = 6.6 hz , 0.6h ) , 3.09 ( dd , j = 12.0 , 11.4 hz , 1h ) , 3.053.01 ( m , 1h ) , 2.94 ( dd , j = 12.6 , 4.2 hz , 0.4h ) , 2.88 ( s , 1.2h ) , 2.80 ( s , 1.8h ) , 2.77 ( dd , j = 12.6 , 4.8 hz , 0.6h ) , 2.74 ( s , 1.8h ) , 2.642.58 ( m , 1.8h ) , 2.52 ( dd , j = 13.5 , 11.4 hz , 0.6h ) , 2.452.32 ( m , 1.6h ) , 2.312.23 ( m , 1.6h ) , 2.151.96 ( m , 2.4h ) , 1.961.83 ( m , 3.6h ) , 1.811.69 ( m , 4h ) , 1.62 ( ddd , j = 13.8 , 9.6 , 3.6 hz , 1h ) , 1.561.51 ( m , 1h ) , 1.311.25 ( m , 1h ) , 1.23 ( d , j = 7.2 hz , 1.8h ) , 1.211.17 ( m , 0.4h ) , 1.09 ( d , j = 6.6 hz , 1.2h ) , 1.03 ( t , j = 6.6 hz , 1.8h ) , 1.03 ( d , j = 6.6 hz , 1.8h ) , 1.00 ( d , j = 6.6 hz , 1.2h ) , 0.98 ( d , j = 6.6 hz , 1.2h ) , 0.980.92 ( m , 0.6h ) , 0.87 ( s , 9h ) , 0.84 ( t , j = 7.2 hz , 1.2h ) , 0.57 ( d , j = 6.6 hz , 1.2h ) ppm . c nmr ( 150 mhz , cdcl3 , mixture of rotamers , major and minor ) : 172.7 , 172.1 , 171.9 , 171.2 , 170.7 , 170.5 , 170.3 , 170.2 , 170.0 , 168.6 , 158.8 , 158.7 , 130.7 , 130.6 , 128.7 , 128.5 , 114.2 , 114.0 , 78.1 , 77.6 , 76.2 , 75.8 , 68.6 , 67.3 , 60.7 , 59.9 , 59.5 , 58.1 , 57.9 , 57.4 , 55.5 , 55.4 , 54.1 , 50.9 , 49.8 , 47.8 , 45.0 , 43.9 , 42.9 , 41.0 , 39.8 , 38.8 , 37.6 , 37.5 , 37.4 , 37.0 , 36.9 , 34.1 , 34.1 , 33.2 , 33.0 , 31.5 , 30.6 , 30.6 , 29.3 , 29.3 , 29.0 , 26.2 , 26.2 , 25.7 , 25.6 , 25.3 , 25.2 , 24.8 , 20.9 , 19.9 , 15.1 , 14.3 , 14.1 , 14.1 , 10.0 , 10.0 ppm . hrms ( esi ) m / z calcd for c43h67n5o8sna ( m + na ) 836.4603 , found 836.4607 . yield 6.8 mg , 70% in 3 steps ; [ ]d 59.5 ( c 0.037 , ch2cl2 ) ; tr = 26.2 min . h nmr ( 600 mhz , cdcl3 , mixture of rotamers , major and minor ( 7/3 ) ) : 7.14 ( d , j = 8.4 hz , 0.6h ) , 7.12 ( d , j = 8.4 hz , 1.4h ) , 6.80 ( d , j = 8.4 hz , 1.4h ) , 6.78 ( d , j = 8.4 hz , 0.6h ) , 6.18 ( d , j = 9.0 hz , 0.7h ) , 5.73 ( d , j = 10.2 hz , 0.3h ) , 5.27 ( d , j = 11.4 hz , 0.3h ) , 5.185.11 ( m , 1h ) , 4.96 ( d , j = 12.0 hz , 0.3h ) , 4.89 ( d , j = 10.8 hz , 1h ) , 4.60 ( q , j = 6.0 hz , 0.7h ) , 4.454.37 ( m , 1h ) , 4.354.31 ( m , 0.7h ) , 4.15 ( t , j = 8.4 hz , 0.3h ) , 4.104.06 ( m , 0.7h ) , 3.87 ( d , j = 10.8 hz , 0.7h ) , 3.76 ( s , 3h ) , 3.70 ( t , j = 10.8 hz , 0.7h ) , 3.633.59 ( m , 1h ) , 3.34 ( dd , j = 10.8 , 8.4 hz , 0.3h ) , 3.31 ( q , j = 6.6 hz , 0.3h ) , 3.24 ( dd , j = 10.8 , 8.4 hz , 0.7h ) , 3.12 ( dd , j = 12.6 , 11.4 hz , 0.3h ) , 3.093.04 ( m , 1.4h ) , 2.95 ( dd , j = 12.6 , 3.6 hz , 0.7h ) , 2.88 ( s , 2.1h ) , 2.87 ( s , 0.9h ) , 2.76 ( s , 0.9h ) , 2.752.73 ( m , 0.3h ) , 2.58 ( s , 2.1h ) , 2.41 ( ddd , j = 13.2 , 13.2 , 4.8 hz , 0.7h ) , 2.362.26 ( m , 2.3h ) , 2.232.18 ( m , 1h ) , 2.142.10 ( br m , 0.7h ) , 2.082.02 ( m , 1.3h ) , 1.981.91 ( m , 3.3h ) , 1.891.82 ( m , 2.3h ) , 1.781.73 ( m , 3.3h ) , 1.601.56 ( m , 0.7h ) , 1.51 ( m , 1h ) , 1.291.23 ( m , 2.8h ) , 1.17 ( s , 0.9h ) , 1.16 ( s , 2.1h ) , 1.071.06 ( m , 5.1h ) , 1.03 ( t , j = 7.2 hz , 0.9h ) , 0.98 ( d , j = 6.6 hz , 3h),0.87 ( s , 9h ) , 0.83 ( t , j = 7.2 hz , 2.1h ) , 0.49 ( d , j = 6.6 hz , 2.1h ) ppm . c nmr ( 150 mhz , cdcl3 , mixture of rotamers , major and minor ) : 179.6 , 178.4 , 172.2 , 172.0 , 172.0 , 171.8 , 170.9 , 170.4 , 170.3 , 170.2 , 170.1 , 169.8 , 158.9 , 158.6 , 130.7 , 130.6 , 128.7 , 128.5 , 114.2 , 113.9 , 77.7 , 75.8 , 75.3 , 74.8 , 73.9 , 60.8 , 60.0 , 59.3 , 57.8 , 57.4 , 55.5 , 55.4 , 54.2 , 50.9 , 49.8 , 47.8 , 46.7 , 46.5 , 40.0 , 38.2 , 38.0 , 37.6 , 37.3 , 37.0 , 36.0 , 35.2 , 35.1 , 34.9 , 34.6 , 34.1 , 33.7 , 31.7 , 31.4 , 30.8 , 30.8 , 30.7 , 29.8 , 29.5 , 29.3 , 28.9 , 26.3 , 26.3 , 25.8 , 25.6 , 25.6 , 25.5 , 25.3 , 25.0 , 24.2 , 22.8 , 20.7 , 20.3 , 18.7 , 18.3 , 14.9 , 14.6 , 14.3 , 14.1 , 14.1 , 10.7 , 10.0 ppm . hrms ( esi ) m / z calcd for c45h71n5o8sna ( m + na ) 864.4916 , found 864.4918 . yield 4.3 mg , 45% in 3 steps ; [ ]d 82.6 ( c 0.023 , ch2cl2 ) ; tr = 24.8 min . h nmr ( 600 mhz , cdcl3 , mixture of rotamers , major and minor ( 6/4 ) ) : 7.13 ( d , j = 9.0 hz , 1.2h ) , 7.13 ( d , j = 9.0 hz , 0.8h ) , 6.82 ( d , j = 9.0 hz , 0.8h ) , 6.79 ( d , j = 9.0 hz , 1.2h ) , 6.23 ( d , j = 8.4 hz , 0.4h ) , 5.86 ( d , j = 9.6 hz , 0.6h ) , 5.23 ( d , j = 11.4 hz , 0.6h ) , 5.19 ( ddd , j = 10.5 , 10.5 , 4.8 hz , 0.6h ) , 5.05 ( ddd , j = 10.8 , 8.4 , 4.2 hz , 0.4h ) , 4.95 ( dd , j = 13.2 , 3.0 hz , 0.6h ) , 4.88 ( dd , j = 12.6 , 3.6 hz , 0.4h ) , 4.82 ( d , j = 11.4 hz , 0.4h ) , 4.73 ( d , j = 11.4 hz , 0.6h ) , 4.59 ( q , j = 6.6 hz , 0.4h ) , 4.23 ( qd , j = 8.4 , 4.8 hz , 0.4h ) , 4.364.34 ( m , 0.4h ) , 4.314.23 ( m , 1h ) , 4.23 ( t , j = 7.8 hz , 0.6h ) , 4.19 ( d , j = 10.2 hz , 0.4h ) , 4.134.09 ( m , 0.4h ) , 3.77 ( s , 1.8h ) , 3.77 ( s , 1.2h ) , 3.693.64 ( m , 0.6h ) , 3.643.60 ( m , 0.4h ) , 3.553.51 ( m , 0.4h ) , 3.50 ( dd , j = 10.8 , 7.8 hz , 0.4h ) , 3.46 ( qd , j = 11.1 , 3.6 hz , 0.6h ) , 3.38 ( dd , j = 10.8 , 7.8 hz , 0.6h ) , 3.26 ( q , j = 6.6 hz , 0.6h ) , 3.092.98 ( m , 1.4h ) , 2.94 ( s , 1.2h ) , 2.802.76 ( m , 1.6h ) , 2.74 ( s , 1.8h ) , 2.74 ( s , 1.8h ) , 2.70 ( dd , j = 11.4 , 11.4 hz , 0.6h ) , 2.632.57 ( m , 2.2h ) , 2.44 ( ddd , j = 16.8 , 5.4 , 3.0 hz , 0.6h ) , 2.412.34 ( m , 1h ) , 2.302.22 ( m , 1.2h ) , 2.202.17 ( m , 0.4h ) , 2.162.10 ( m , 1h ) , 2.092.03 ( m , 1.4h ) , 1.981.84 ( m , 2.4h ) , 1.79 ( td , j = 13.2 , 3.6 hz , 0.6h ) , 1.751.61 ( m , 4.4h ) , 1.581.52 ( m , 0.6h ) , 1.49 ( td , j = 11.4 , 3.6 hz , 0.6h ) , 1.33 ( ddd , j = 13.8 , 11.1 , 3.0 hz , 0.4h ) , 1.291.26 ( m , 1.4h ) , 1.23 ( d , j = 7.2 hz , 1.8h ) , 1.121.09 ( m , 2.2h ) , 1.07 ( d , j = 6.6 h , 1.8h ) , 1.05 ( d , j = 7.2 hz , 1.8h ) , 1.01 ( t , j = 7.2 hz , 1.8h ) , 0.970.95 ( m , 3.6h ) , 0.92 ( m , 0.4h ) , 0.87 ( s , 5.4h ) , 0.87 ( s , 3.6h ) , 0.85 ( t , j = 7.2 hz , 1.2h ) , 0.43 ( d , j = 6.6 hz , 1.2h ) ppm . c nmr ( 150 mhz , cdcl3 , mixture of rotamers , major and minor ) : 175.3 , 174.9 , 172.6 , 172.2 , 171.9 , 171.4 , 171.1 , 170.7 , 170.5 , 170.5 , 170.3 , 170.0 , 158.9 , 158.7 , 130.7 , 130.5 , 128.7 , 128.4 , 114.3 , 114.0 , 78.0 , 77.6 , 75.5 , 74.9 , 72.5 , 72.1 , 60.7 , 59.8 , 59.3 , 58.3 , 56.9 , 55.5 , 55.4 , 53.8 , 51.6 , 49.6 , 49.0 , 47.8 , 47.5 , 40.0 , 39.4 , 38.3 , 38.0 , 37.8 , 37.6 , 37.5 , 37.1 , 36.8 , 35.2 , 35.2 , 34.2 , 34.0 , 33.2 , 32.5 , 31.6 , 31.1 , 30.8 , 30.5 , 29.3 , 29.2 , 28.8 , 26.2 , 25.8 , 25.7 , 25.3 , 25.2 , 24.8 , 24.7 , 20.6 , 20.1 , 16.5 , 16.4 , 14.6 , 14.3 , 14.0 , 14.0 , 10.1 , 9.9 ppm . hrms ( esi ) m / z calcd for c44h69n5o8sna ( m + na ) 850.4759 , found 850.4773 . human colon adenocarcinoma hct116 cells were purchased from atcc ( manassas , va ) and cultured in dulbecco s modified eagle s medium ( invitrogen , carlsbad , ca ) supplemented with 10% fetal bovine serum ( hyclone , logan , ut ) at 37 c humidified air and 5% co2 . hct116 cells were seeded at a density of 1 10 cells per well in 96-well plates . then 24 h later , the cells were treated with various concentrations of apratoxins or solvent control ( 1% etoh ) . after 48 h of incubation , cell viability was detected using mtt according to the manufacturer s instructions ( promega , madison , wi ) . hct116 cells ( 1 10 cells per well ) were seeded in 96-well plates and one day later treated with various concentrations of apratoxins or solvent control . after 12 h of incubation , culture supernatants were collected for detection of vegf - a by using an alphalisa kit ( perkinelmer , waltham , ma ) following the manufacturer s instructions . briefly , acceptor bead and anti - vegf antibody were incubated first with the supernatants for 60 min , donor beads were added later and incubated for another 30 min , and then vegf - a levels were detected using envision ( perkinelmer ) . hct116 cells were seeded in 6-well plates at a density of 4 10 cells and the next day treated with various concentrations of apratoxins or solvent control ( 1% etoh ) . then 24 h later , whole cell lysates were collected using phosphosafe buffer ( emd chemicals , inc . , gibbstown , nj ) . the protein concentration was measured with the bca protein assay kit ( thermo fisher scientific , rockford , il ) . lysates containing equal amounts of protein were separated by sds polyacrylamide gel electrophoresis ( 412% ) , transferred to polyvinylidene difluoride membranes , probed with primary and secondary antibodies , and detected with the supersignal west femto maximum sensitivity substrate ( thermo fisher scientific ) . anti - met , anti - vegfr2 , and secondary antimouse antibodies were from cell signaling technology , inc . three five week old female nude mice ( nu / nu ) were obtained from charles river laboratory ( wilmington , ma ) . one 10 hct116 cells in a volume of 100 l of sterile saline were injected subcutaneously on the left rear flank of a nude mouse to establish tumors . tumor dimensions were measured using calipers every day , and tumor volumes were calculated using the formula w l 0.5 , where width ( w ) length ( l ) . tumors with a starting volume bigger than 100 mm were excluded from the analysis . mice were injected intraperitoneally with the doses of 2 g / mouse ( 0.1 mg / kg ) , 5 g / mouse ( 0.25 mg / kg ) of 1c or solvent ( dmso ) control every day ( 25 l ) until the tumor size in one dimension reached 15 mm and tumor tissue was harvested on the following day . finally , 50 mg of tumor tissue was sonicated in phosphosafe lysis buffer ( emd chemicals , inc . ) and used for immunoblot analysis described as the above . all studies were carried out under the protocol approved by the institutional animal care and use committee at the university of florida . hplc - ms was done on a 3200 qtrap ( applied biosystems ) equipped with a shimadzu ( kyoto , japan ) uflc system . pooled cd1 mouse liver ( female ) microsomes were purchased from xenotech , lcc ( lenexa , ks ) with protein concentrations of 0.5 mg / ml . cell lysates were prepared with phosphosafe lysis buffer ( novagen , san diego , ca ) . stock solutions of 1a d were prepared by dissolving the compounds in ethanol to give a 1 mg / ml solution . aliquots of this stock solution were then obtained to afford a 40 g / ml solution in acetonitrile . serial dilution of the 40 g / ml solution in acetonitrile gave standard solutions with concentrations of 25 , 12.5 , 2.5 , 1.25 , 0.25 , 0.125 , 0.025 , and 0.0125 g / ml . a 1 mg / ml stock solution of the internal standard harmine was prepared in ethanol , which subsequently was used to prepare a 100 g / ml solution with ethanol . an aliquot of the 100 g / ml harmine solution was diluted to 35 ng / ml with ethyl acetate to serve as the working internal standard solution . in vitro plasma stability of 1a first , 10 l of apratoxins ( 25 g / ml ) were added to 100 l of mouse serum , and the solution was then vortex - mixed for 15 s and incubated for 0.25 min to 24 h. at the end of each incubation period , 400 l of ethyl acetate was added to each tube , followed by 200 l of harmine to quench the reaction and extract remaining apratoxin . samples were further incubated in a thermomixer at 27 c ( 750 rpm , 5 min ) and later centrifuged for 5 min at 1643 g . a volume of 10 l of the reconstituted solution was injected into the hplc - ms system . the stability of 1a d in the presence of mouse microsomes was determined by using an adaptation of a published procedure . in brief , microsomes were added to prewarmed phosphate buffer ( 100 mm , ph 7.4 ) at 37 c . apratoxins ( 3 l ) were added to the microsomal preparation followed by nadph cofactor solution ( 1.3 mm nadp , 3.3 mm glucose 6-phosphate , 0.4 u / ml glucose-6-phosphate dehydrogenase , 3.3 mm mgcl2 ) . the reaction was allowed to proceed for 3 min to 2 h at 37 c ( thermomixer , 1050 rpm ) . the reaction was quenched by addition of ethyl acetate and subsequently spiked with harmine . the zero time point was defined by denaturing the microsomes with ethyl acetate before the addition of apratoxins . incubation of apratoxins with microsomes alone was also performed following the same procedure to determine nadph - independent metabolism . the final concentration of the incubation mixture contained 0.5 mg / ml protein concentration and 1 m apratoxins . aliquots of hct116 cell lysates were diluted with 25 mm tris - hcl buffer , ph 8.0 , to give a final reaction volume of 100 l and protein concentration of 0.7 mg / ml . cell lysate solutions were incubated with 10 l of apratoxins ( 25 g / ml ) for 0.25 min to 24 h. remaining apratoxins were extracted from the reaction solution at the end of the incubation periods with ethyl acetate using the same procedure as described for the plasma stability assay . the stability of 1a d in aqueous solution was determined in 100 mm phosphate buffer , ph 4.88 , 100 mm phosphate buffer , ph 7.4 . portions of each solution ( 100 l ) were spiked with 10 l of apratoxin solution ( 25 g / ml ) and allowed to incubate for 0.25 min to 24 h. the reaction was quenched at the end of each time point , and the remaining apratoxins were extracted with ethyl acetate , as in the plasma stability study . analysis of 1a d was done by using hplc - ms [ column , onyx monolithic c18 ( 3.0 mm 100 mm ) , phenomenex ( torrance , ca ) ; solvent , water ( solvent a ) acetonitrile ( solvent b ) ; flow rate , 0.5 ml / min ; detection by electrospray ionization ms in positive ion mode ( mrm scan ) ] . a stepwise gradient elution was used starting at 60% b and 40% a , then increasing to 80% b at 5 min and maintained at this condition for 5 min . parameters were optimized before analysis by using direct syringe infusion . the retention times ( tr , min ; mrm ion pair ) of the analytes and internal standard are as follows : harmine ( 2.2 ; 213.1 169.9 ) , 1a ( 4.1 ; 828.5 432.2 ) , 1b ( 4.05 ; 814.5 418.2 ) , 1c ( 5.2 ; 842.5 446.2 ) , 1d ( 4.9 ; 828.5 432.2 ) . compound - dependent parameters used were as follows : apratoxins , declustering potential ( dp ) 51 , entrance potential ( ep ) 12 , collision energy ( ce ) 45 , collision cell exit potential ( cxp ) 6 , collision cell entrance potential ( cep ) 32 ; and harmine , dp 56.0 , ep 4.5 , ce 44.0 , cxp 5 , cep 16.0 . source gas parameters used were as follows : curtain gas , 15.0 ; collision gas low , ionspray voltage 5500 ; temperature , 600.0 ; ion source gas 1 50.0 ; ion source gas 2 60.0 . calibration curves for 1a d in the presence of mouse serum , hct116 cell lysates , and aqueous solutions were generated by least - squares linear regression analysis of the analyte peak area and internal standard peak area ratio against the nominal concentration of the standard solutions . a linear regression analysis was performed , and the concentration of remaining apratoxins at each time point was determined through interpolation for plasma , cellular , and aqueous stability experiments . the amount of remaining apratoxins with microsome incubation was determined from the peak area ratio of apratoxins at tx ( 3 min to 2 h ) and t0 . all calculations were done by using analyst 1.4.2 ( applied biosystems ) quantitate mode . human colon adenocarcinoma hct116 cells were purchased from atcc ( manassas , va ) and cultured in dulbecco s modified eagle s medium ( invitrogen , carlsbad , ca ) supplemented with 10% fetal bovine serum ( hyclone , logan , ut ) at 37 c humidified air and 5% co2 . hct116 cells were seeded at a density of 1 10 cells per well in 96-well plates . then 24 h later , the cells were treated with various concentrations of apratoxins or solvent control ( 1% etoh ) . after 48 h of incubation , cell viability was detected using mtt according to the manufacturer s instructions ( promega , madison , wi ) . hct116 cells ( 1 10 cells per well ) were seeded in 96-well plates and one day later treated with various concentrations of apratoxins or solvent control . after 12 h of incubation , culture supernatants were collected for detection of vegf - a by using an alphalisa kit ( perkinelmer , waltham , ma ) following the manufacturer s instructions . briefly , acceptor bead and anti - vegf antibody were incubated first with the supernatants for 60 min , donor beads were added later and incubated for another 30 min , and then vegf - a levels were detected using envision ( perkinelmer ) . hct116 cells were seeded in 6-well plates at a density of 4 10 cells and the next day treated with various concentrations of apratoxins or solvent control ( 1% etoh ) . then 24 h later , whole cell lysates were collected using phosphosafe buffer ( emd chemicals , inc . , gibbstown , nj ) . the protein concentration was measured with the bca protein assay kit ( thermo fisher scientific , rockford , il ) . lysates containing equal amounts of protein were separated by sds polyacrylamide gel electrophoresis ( 412% ) , transferred to polyvinylidene difluoride membranes , probed with primary and secondary antibodies , and detected with the supersignal west femto maximum sensitivity substrate ( thermo fisher scientific ) . anti - met , anti - vegfr2 , and secondary antimouse antibodies were from cell signaling technology , inc . three five week old female nude mice ( nu / nu ) were obtained from charles river laboratory ( wilmington , ma ) . one 10 hct116 cells in a volume of 100 l of sterile saline were injected subcutaneously on the left rear flank of a nude mouse to establish tumors . tumor dimensions were measured using calipers every day , and tumor volumes were calculated using the formula w l 0.5 , where width ( w ) length ( l ) . tumors with a starting volume bigger than 100 mm were excluded from the analysis . mice were injected intraperitoneally with the doses of 2 g / mouse ( 0.1 mg / kg ) , 5 g / mouse ( 0.25 mg / kg ) of 1c or solvent ( dmso ) control every day ( 25 l ) until the tumor size in one dimension reached 15 mm and tumor tissue was harvested on the following day . finally , 50 mg of tumor tissue was sonicated in phosphosafe lysis buffer ( emd chemicals , inc . ) and used for immunoblot analysis described as the above . all studies were carried out under the protocol approved by the institutional animal care and use committee at the university of florida . hplc - ms was done on a 3200 qtrap ( applied biosystems ) equipped with a shimadzu ( kyoto , japan ) uflc system . mouse serum and harmine pooled cd1 mouse liver ( female ) microsomes were purchased from xenotech , lcc ( lenexa , ks ) with protein concentrations of 0.5 mg / ml . hct116 cell lysates were prepared with phosphosafe lysis buffer ( novagen , san diego , ca ) . stock solutions of 1a d were prepared by dissolving the compounds in ethanol to give a 1 mg / ml solution . aliquots of this stock solution were then obtained to afford a 40 g / ml solution in acetonitrile . serial dilution of the 40 g / ml solution in acetonitrile gave standard solutions with concentrations of 25 , 12.5 , 2.5 , 1.25 , 0.25 , 0.125 , 0.025 , and 0.0125 g / ml . a 1 mg / ml stock solution of the internal standard harmine was prepared in ethanol , which subsequently was used to prepare a 100 g / ml solution with ethanol . an aliquot of the 100 g / ml harmine solution was diluted to 35 ng / ml with ethyl acetate to serve as the working internal standard solution . in vitro plasma stability of 1a first , 10 l of apratoxins ( 25 g / ml ) were added to 100 l of mouse serum , and the solution was then vortex - mixed for 15 s and incubated for 0.25 min to 24 h. at the end of each incubation period , 400 l of ethyl acetate was added to each tube , followed by 200 l of harmine to quench the reaction and extract remaining apratoxin . samples were further incubated in a thermomixer at 27 c ( 750 rpm , 5 min ) and later centrifuged for 5 min at 1643 g . a volume of 10 l of the reconstituted solution was injected into the hplc - ms system . the stability of 1a d in the presence of mouse microsomes was determined by using an adaptation of a published procedure . in brief , microsomes were added to prewarmed phosphate buffer ( 100 mm , ph 7.4 ) at 37 c . apratoxins ( 3 l ) were added to the microsomal preparation followed by nadph cofactor solution ( 1.3 mm nadp , 3.3 mm glucose 6-phosphate , 0.4 u / ml glucose-6-phosphate dehydrogenase , 3.3 mm mgcl2 ) . the reaction was allowed to proceed for 3 min to 2 h at 37 c ( thermomixer , 1050 rpm ) . the reaction was quenched by addition of ethyl acetate and subsequently spiked with harmine . the zero time point was defined by denaturing the microsomes with ethyl acetate before the addition of apratoxins . incubation of apratoxins with microsomes alone was also performed following the same procedure to determine nadph - independent metabolism . the final concentration of the incubation mixture contained 0.5 mg / ml protein concentration and 1 m apratoxins . aliquots of hct116 cell lysates were diluted with 25 mm tris - hcl buffer , ph 8.0 , to give a final reaction volume of 100 l and protein concentration of 0.7 mg / ml . cell lysate solutions were incubated with 10 l of apratoxins ( 25 g / ml ) for 0.25 min to 24 h. remaining apratoxins were extracted from the reaction solution at the end of the incubation periods with ethyl acetate using the same procedure as described for the plasma stability assay . the stability of 1a d in aqueous solution was determined in 100 mm phosphate buffer , ph 4.88 , 100 mm phosphate buffer , ph 7.4 . portions of each solution ( 100 l ) were spiked with 10 l of apratoxin solution ( 25 g / ml ) and allowed to incubate for 0.25 min to 24 h. the reaction was quenched at the end of each time point , and the remaining apratoxins were extracted with ethyl acetate , as in the plasma stability study . analysis of 1a d was done by using hplc - ms [ column , onyx monolithic c18 ( 3.0 mm 100 mm ) , phenomenex ( torrance , ca ) ; solvent , water ( solvent a ) acetonitrile ( solvent b ) ; flow rate , 0.5 ml / min ; detection by electrospray ionization ms in positive ion mode ( mrm scan ) ] . a stepwise gradient elution was used starting at 60% b and 40% a , then increasing to 80% b at 5 min and maintained at this condition for 5 min . the retention times ( tr , min ; mrm ion pair ) of the analytes and internal standard are as follows : harmine ( 2.2 ; 213.1 169.9 ) , 1a ( 4.1 ; 828.5 432.2 ) , 1b ( 4.05 ; 814.5 418.2 ) , 1c ( 5.2 ; 842.5 446.2 ) , 1d ( 4.9 ; 828.5 432.2 ) . compound - dependent parameters used were as follows : apratoxins , declustering potential ( dp ) 51 , entrance potential ( ep ) 12 , collision energy ( ce ) 45 , collision cell exit potential ( cxp ) 6 , collision cell entrance potential ( cep ) 32 ; and harmine , dp 56.0 , ep 4.5 , ce 44.0 , cxp 5 , cep 16.0 . source gas parameters used were as follows : curtain gas , 15.0 ; collision gas low , ionspray voltage 5500 ; temperature , 600.0 ; ion source gas 1 50.0 ; ion source gas 2 60.0 . calibration curves for 1a d in the presence of mouse serum , hct116 cell lysates , and aqueous solutions were generated by least - squares linear regression analysis of the analyte peak area and internal standard peak area ratio against the nominal concentration of the standard solutions . a linear regression analysis was performed , and the concentration of remaining apratoxins at each time point was determined through interpolation for plasma , cellular , and aqueous stability experiments . the amount of remaining apratoxins with microsome incubation was determined from the peak area ratio of apratoxins at tx ( 3 min to 2 h ) and t0 . all calculations were done by using analyst 1.4.2 ( applied biosystems ) quantitate mode .

key clinical messagesarcoidosis is a diagnosis that should be considered in patients receiving interferon therapy , who present with anemia and multiorgan dysfunction regardless of the duration of their treatment . when sarcoidosis is suspected , bone marrow biopsy should be considered especially for cases predominant by extrapulmonary features . apratoxins are cytotoxic natural products originally isolated from marine cyanobacteria that act by preventing cotranslational translocation early in the secretory pathway to downregulate receptor levels and inhibit growth factor secretion , leading to potent antiproliferative activity . through rational design and total synthesis of an apratoxin a / e hybrid , apratoxin s4 ( 1a ) , we have previously improved the antitumor activity and tolerability in vivo . compound 1a and newly designed analogues apratoxins s7s9 ( 1b d ) , with various degrees of methylation at c34 ( 1b , c ) or epimeric configuration at c30 ( 1d ) , were efficiently synthesized utilizing improved procedures . optimizations have been applied to the synthesis of key intermediate aldehyde 7 and further include the application of leighton s silanes and modifications of kelly s methods to induce thiazoline ring formation in other crucial steps of the apratoxin synthesis . apratoxin s9 ( 1d ) exhibited increased activity with subnanomolar potency . apratoxin s8 ( 1c ) lacks the propensity to be deactivated by dehydration and showed efficacy in a human hct116 xenograft mouse model .

seizure in patients with systemic lupus erythematosus ( sle ) is considered to be one of the major manifestations of the disease . the prevalence of seizure has been documented as about 10 to 20% in the patients with sle ( 1 , 2 ) . this neuropsychiatric event is likely to be primary to the sle or secondary due to other concomitant conditions , including infection , hypertension , hyperlipidemia , metabolic derangement and the toxic effects of therapy ( 3 , 4 ) . hyperglycemic hyperosmolar syndrome ( hhs ) is a metabolic emergency , and it commonly occurs in elderly , disabled people with type 2 diabetes . the report of sament and schwartz in 1957 is widely credited as the original description of hhs . the proposed criteria for hhs are a plasma glucose concentration > 33.3 mm / l , a serum carbon dioxide concentration > 15 mm / l , no or mild ketonuria , absent to mild ketonemia , a serum osmolarity > 320 mm / kg and mental change ( 5 ) . however , hhs without other metabolic derangement and that occurs in a patient with lupus nephritis without diabetes and who received short term steroid therapy is a very rare complication . a 51-yr - old female was referred to our outpatient clinic for the evaluation of generalized edema on november 17 , 2009 . two years previously , she had been diagnosed with idiopathic thrombocytopenic purpura ( itp ) . she had taken no medicine . except for the itp , she had no history of systemic disease like diabetes or hypertension . on admission , her blood pressure was 140/80 mmhg with a pulse rate of 76 beats / min . the chest radiography revealed cardiomegaly ( the ct ratio was 0.54 ) and bilateral pleural effusions . both kidneys showed diffusely increased parenchymal echogenicity with partially obliteration of the corticomedullary differentiation and a normal range of size ( the right kidney was 10.3 cm by 4.5 cm in size , and the left kidney was 9.5 cm by 4.5 cm in size ) . the laboratory findings showed nephrotic syndrome with generalized edema ; the 24 hr urine protein was 5.3 g/24 hr , the total cholesterol was 7 mm / l ( normal : < 5.2 mm / l ) and the serum albumin was 22 the fasting blood glucose and hemoglobin a1c ( hba1c ) were 5.4 mm / l and 0.05 hb fraction , respectively . the blood urea nitrogen and serum creatinine were 20.6 mm / l ( normal : 2.6 - 7.3 mm / l ) and 0.23 mm / l ( normal : 0.05 - 0.11 in addition , the patient had a positive antinuclear antibody titer at 1:1,600 ( normal : negative ) , positive antibodies against double - stranded dna at 400 iu / ml ( normal : < 100 iu / ml ) and she was negative for antiphospholipid antibodies . she was diagnosed with systemic lupus erythematosus by the american college of rheumatology criteria ( 6 ) . at diagnosis , a renal biopsy was performed . that showed diffuse global glomerulonephritis with active lesion and iv - g(a ) by the isn / rps classification ( 7 ) . immunosuppressions consisting of high - dose steroid ( methylprednisolone 750 mg for 3 days followed by prednisolone 60 mg ) , cyclophosphamide pulses ( 500 mg at the 6th and 36th day of admission ) , as well as plasma exchange were all started . in addition , she had oliguria that was refractory to diuretics , and this led us to start hemodialysis . while maintaining a stable clinical course , a generalized myoclonic seizure occurred at the 47th day of admission . at that time , her body temperature was 36.1. the laboratory and neurology studies showed hyperglycemic hyperosmolar syndrome with a blood glucose level of > 44.4 mm / l , the serum osmolarity was 382 mm / kg ( normal : 275 - 300 mm / kg ) and the hemoglobin a1c was 0.082 hb fraction . the serum ph was 7.348 and the bicarbonate was 26.2 mm / l ( normal : 21 - 28 the c - reactive protein was normal at 3.9 mg / l ( normal : 0.1 - 4.7 mg / l ) . g / l ) , respectively . at shortly before seizure , the sledai score was 12 . the seizure stopped after the blood sugar and serum osmolarity declined below the upper normal limit mentioned above . the patient became asymptomatic and she was discharged 10 weeks after admission under maintenance therapy with prednisolone 10 mg , insulin glargine 12 unit and nateglinide 270 mg . on follow - up 1 , 2 , and 3 month after discharge , the hba1c was 0.051 , 0.049 , and 0.06 hb fraction , respectively . 1 , 2 . the patient remained asymptomatic under maintenance therapy with deflazacort 12 mg and without insulin or medication for blood sugar control . in patients with lupus nephritis , the causes of seizure are a multiplicity of different factors , such as cns involvement of sle , infections , hypertension and metabolic derangement . a large prospective study reported that infection was the most common cause of neurologic episodes and primary involvement of sle was the secondary cause ( 8) . the presence of seizure increases the risk of death in patients with sle by approximately 2-fold ( 2 , 9 ) . when seizure is noted in patients with lupus nephritis , identifying the cause and proper treatment are important to improve survival . most studies have revealed that reversible posterior leukoencephalopathy syndrome and cerebral vascular accident are other important causes of seizure in patients with lupus nephritis . by contrast , the neurologic episodes by purely metabolic derangement without infection or cns lupus are very rare , and especially hhs was a seldom cause of seizure ( 8) . steroid is associated with the risk of hyperglycemia in patients with or without diabetes ( 10 , 11 ) . this inhibit insulin signalling in skeletal muscle and liver , resulting in reduced glucose uptake and glycogen systhesis , increased breakdown of skeletal muscle mass , increased hepatic glucose production . in additions , steroid increase whole body lipolysis , resulting in increased nonesterified fatty acids ( nefa ) and triglyceride ( tg ) . augmented levels of nfea and tg may enhance the accumulation of intracellular lipids , which impair glucose metabolisms and induce hepatic steatosis , dyslpidemia . steroid most likely exert inhibitory effects in the beta - cell after exposure , resulting decreased beta - cell insulin production ( 12 - 14 ) . as an acute complication of diabetes , the overall mortality of hhs is in the range of 10%-50% . however , when therapy is promptly instituted , the mortality rate can be reduced . treatment is targeted towards 1 ) correcting the intravascular volume and electrolyte derangements ; 2 ) decreasing the hyperglycemia and hyperosmolarity by insulin ; and 3 ) identifying and managing the comorbidities triggering the hhs ( 5 , 16 ) . althought hyperglycemia due to steroid therapy is a common , but acute complication of diabetes , hhs as a first manifestation is a very rare event . our case showed that seizure developed due to hhs despite of the short - term steroid therapy . we should consider hhs as a cause of seizure in patient with lupus nephritis and who are treated with steroid . in conclusion , our case highlights the importance of regular blood glucose testing during or before high - dose steroid therapy . stress urinary incontinence ( sui ) , pelvic organ prolapse , and chronic pelvic pain are frequent complaints of women in perimenopause and menopause and result mainly from pelvic floor insufficiency . proper statics of the pelvic floor provides adequate support for the urethra and bladder , which determines the correct mechanism of continence . anatomical disorders such as loss of support for the bladder base and the proximal urethra are a direct cause of sui.13 women diagnosed with sui demonstrated changed configuration of these structures as well as disorders in their functioning . among the causes of these changes are the weakening of connective tissue structures and the loosening of connections within the pelvic floor.47 it is believed that the menopause plays the most significant role in this pathogenesis . symptoms related to the urogenital system may affect 50% of women in this period.3,813 many authors3,9,12,1417 indicate a decreased level of estrogens as the main cause of the symptoms . one of the key roles of estrogens , which may be impaired during menopause , is their influence on the synthesis and metabolism of collagen , eg , within the lower urinary tract , and on the increase in the number of muscle fibers of the detrusor muscle as well as other muscles comprising the pelvic floor.3,1416 during the menopause , a reduced number of estrogen receptors in the epithelia of the urethra , bladder triangle , or vaginal mucosa can be observed . this decrease is also seen in the structures supporting the pelvic organs , eg , uterosacral ligament , levator ani muscle , and pubocervical fascia.11,15,1821 the other cause of disorders in the statics of the pelvic floor is damage to the muscle and connective tissue structures due to vaginal deliveries.11,2224 botelho et al24 reported a significant loss of muscle contractility during electromyography ( emg ) evaluation in women who underwent vaginal delivery . in patients who had undergone cesarean section , a decrease in muscle function was not observed . it should be stated that despite many studies on the negative impact of episiotomy on the pelvic floor , in many countries , it is still a routine practice . a study conducted by blondel et al25 based on the euro - peristat project demonstrated that many european countries had episiotomy rates > 60% ( poland 67.5% , romania 68.2% , portugal 72.9% , and cyprus 75% ) . recent randomized clinical trials have shown that currently the most comprehensive therapeutic procedure in sui are properly chosen and properly executed pelvic floor muscle ( pfm ) exercises along with the use of objective , minimally invasive and safe diagnostic methods ( eg , emg).24,26,27 in physiotherapeutic practice , special attention is being given to the reciprocal anatomical , physiological , and biomechanical relationship of the pelvis and the structures connected to it . the lack of information about these relations from the practical aspect as well as the paucity of scientific papers31,3335 on the impact of posture changes on the pelvic floor and the function of the lower urinary tract prompted this study . the authors attempted to answer the question of whether the position of the lower limbs , affecting the different orientations of the pelvis , can influence pfm activity . the primary aim of this study was to compare the resting and functional bioelectrical activities of pfm depending on three different positions of the lower limbs ( position a , b , c ) in the supine position . the secondary goal was to compare the surface electromyography ( semg ) activity of selected synergists of the pfm , eg , adductor magnus ( am ) , rectus abdominis ( ra ) , and gluteus maximus ( gm ; two sides : left and right ) , in different positions of the lower limbs in the supine position as well as to determine the relationship between the myoelectric activity of the pfm and synergist muscles . this was a prospective , cross - sectional observational study evaluating resting and functional activities of the pfm depending on the position of the lower limbs in menopausal women . the presented study is a part of the research project funded by the national science centre in the preludium call on the basis of the decision number dec 2011/03/n / nz7/00505 . the study was approved by the bioethics committee of the wroclaw medical university on 5 july 2012 with the approval number kb 611/2012 . moreover , the study was registered at the australian new zealand clinical trials registry platform ( actrn1261300114470736 ) as a prospective observational study . the study was carried out at the department and clinic of urology of university hospital in wroclaw between december 2012 and december 2014 . all recruited women were subjected to evaluation by the inclusion and exclusion criteria in order to qualify for the appropriate study group . the inclusion criteria assumed in the study included written informed consent , doctor s and/or physiotherapist s permission to participate in the study , the overall well - being on the day of examination and no contraindications for the semg measurements . the exclusion criteria included age older than 75 years ; no history of menopause ; gynecological surgery surgery in the abdomen , pelvis , and lower extremities in the last 10 years ; injuries to the lower extremities , pelvis or , spine on the examination day ; contraindications for measurements infection , menstruation , and allergies to nickel ; the occurrence of pain during the examination and withdrawing during the examination . the visit protocol with the participants included an interview , instruction on the purpose of measurement and testing procedures and semg measurement of the pfms and their synergists in three positions of lower limbs . the measurements were performed with the lower limbs in three different positions ( figure 1 ) : position a ( position of lower limbs in 90 of flexion in hip and knee joints ; the limbs were placed freely on adequately profiled wedges decreased anterior pelvic tilt ) , position b ( position of lower limbs with a slight flexion of hip and knee joints ; intermediate pelvic position ) and position c ( supine position ; lower limbs without flexion in hip and knee joints and upper limbs resting freely on the couch increased anterior pelvic tilt ) . the order of the positions was established randomly for each participant by the random integer generator ( www.random.org ) . the participants , after setting the pelvis in a particular position , made five 5-second maximal contractions of the pfm ( functional semg activity ) with a 5-second rest between contractions ( resting semg activity ) . the resting and functional bioelectric activities were measured using the myosystem 1400l ( noraxon , scottsdale , az , usa ) with eight channels , along with the surface and endovaginal electrodes . the specification of the device fulfills the requirements set out in isek ( international society of electrophysiology and kinesiology ) and seniam ( surface electromyography for the non - invasive assessment of muscle ) publications . the equipment for recording bioelectric potentials includes main unit with a built - in analog to digital converter ( adc ) card ; signal amplifiers and preamplifiers ; a computer with myoresearch xp master edition software , version 1.04 ; compatible surface and vaginal electrodes . the measuring set was characterized with the semg recording frequency in the range of 10450 hz . the frequency range for the amplifier was 10 hz for high - pass cutoff , with the 500 hz filter for the low - pass cutoff . the level of common mode rejection amounted to a minimum of 100 db , and input impedance for emg channels was > 100 m. the system was characterized by the high sensitivity of the recorded emg signal 1 v . to record the semg signal from the pfm , a pear - shaped endovaginal electrode life - care vaginal probe pr-02 ( everyway medical instruments co. , ltd . the physical examination was conducted with the use of standardized assessment scales for menopause and the symptoms of urinary incontinence . tulsa , ok , usa ) under the license of the medical university in wroclaw . for the measurable variables , the arithmetic mean , all quantitative variables were tested with the shapiro wilk test to determine the type of distribution . comparisons of results between the values obtained in the a , b , and c positions were performed using a parametric test , one - way analysis of variance ( anova ) along with post hoc testing ( tukey s test ) or the nonparametric anova kruskal wallis analysis and multiple comparisons of mean ranks , depending on the fulfillment of the test assumptions . a multivariate linear regression analysis was used to assess relationships between the bioelectrical activity of pfm and bioelectrical activity of all tested synergists ( am , ra , gm for all comparisons , a level of = 0.05 was assumed , and the obtained p - values were rounded to four decimal places . a total of 55 women , aged between 50 and 75 years ( x=64.9 years ; sd = 5.3 years ) , participated in the study . all women underwent the menopause between 40 and 60 years of age ( x=50.7 years ; sd = 5.0 years ) . each participant in the mrs questionnaire reported symptoms typical for the menopause and postmenopause periods . the results of the mrs questionnaire ranged between 1 and 23 points ( x=10.9points ; sd = 5.7 points ) . in position a , the average resting semg activity of the pfm was 6.9 v ( min max : 3.315.3 v ; sd = 2.6 v ) ; in position b also , the result was 6.9 v ( min max : 3.014.8 v ; sd = 2.5 v ) ; and in position c , the resting semg activity was 5.7 v ( min max : 2.611.7 v ; sd = 1.8 v ) . the results of the functional bioelectrical activity of the pfm were as follows : position a 20.3 v ( min max : 4.555.1 v ; sd = 11.8 v ) , position b 19.9 v ( min max : 5.848.2 v ; sd = 10.6 v ) , and position c 25.3 v ( min max : 10.063.8 v ; sd = 10.9 v ) . the highest bioelectrical potential of am ( left and right sides ) , both during the resting and functional activities of the pfm , was obtained in position b. in the case of ra semg activity , there were no statistically significant differences between the results obtained in all positions . the highest semg of the gm ( on the right side ) during the resting and functional activities of the pfm was observed in positions a and b. on the left side , there were no statistically significant differences between positions . multivariate linear regression did not find any association between the resting bioelectrical activity of the pfm and the bioelectrical activity of all synergist muscles in each position ( table 5 ) . similar results were registered taking into account the functional bioelectrical activity of the pfm ( table 6 ) . a total of 55 women , aged between 50 and 75 years ( x=64.9 years ; sd = 5.3 years ) , participated in the study . all women underwent the menopause between 40 and 60 years of age ( x=50.7 years ; sd = 5.0 years ) . each participant in the mrs questionnaire reported symptoms typical for the menopause and postmenopause periods . the results of the mrs questionnaire ranged between 1 and 23 points ( x=10.9points ; sd = 5.7 points ) . in position a , the average resting semg activity of the pfm was 6.9 v ( min max : 3.315.3 v ; sd = 2.6 v ) ; in position b also , the result was 6.9 v ( min max : 3.014.8 v ; sd = 2.5 v ) ; and in position c , the resting semg activity was 5.7 v ( min max : 2.611.7 v ; sd = 1.8 v ) . the results of the functional bioelectrical activity of the pfm were as follows : position a 20.3 v ( min max : 4.555.1 v ; sd = 11.8 v ) , position b 19.9 v ( min max : 5.848.2 v ; sd = 10.6 v ) , and position c 25.3 v ( min max : 10.063.8 v ; sd = 10.9 v ) . the highest bioelectrical potential of am ( left and right sides ) , both during the resting and functional activities of the pfm , was obtained in position b. in the case of ra semg activity , there were no statistically significant differences between the results obtained in all positions . the highest semg of the gm ( on the right side ) during the resting and functional activities of the pfm was observed in positions a and b. on the left side , there were no statistically significant differences between positions . multivariate linear regression did not find any association between the resting bioelectrical activity of the pfm and the bioelectrical activity of all synergist muscles in each position ( table 5 ) . similar results were registered taking into account the functional bioelectrical activity of the pfm ( table 6 ) . the primary aim of the study was to compare the resting and functional bioelectrical activities of pfm in different positions . it was shown that in the supine position ( c ) , the resting semg activity of the pfm reached the lowest values ( 5.7 v ) . both in the position with lower limbs placed on wedges ( a ) and with lower limbs slightly bent ( b ) , the resting activity achieved the same values ( 6.9 v ) . the results confirm the use of the supine position as the most conducive to pfm relaxation . it would seem that this phenomenon may indirectly result from the relaxation of synergist muscles , especially the am and gm ( in the supine position , their insertions are relatively close , which contributes to lower resting potential ) , but the authors did not report any association between pfm and the synergists . the literature reveals only few research studies related to the relationship between posture and the pfm activity . they conducted the measurement of resting pressure , maximum squeeze pressure , and holding periods in seconds , both in supine and standing positions . although they noted significantly higher vaginal resting pressure in the standing position , there were no differences between these two positions in maximal strength and holding time . in the present study , therefore , it seems that this position is justifiably the most commonly used for both the diagnosis and treatment of sui.37 the relationship between the position and the pfm was also assessed by chen et al.35 they evaluated 39 incontinent women to determine the changes in pfm activity during various pelvic tilt angles created by horizontal , dorsiflexed , and plantar - flexed ankle positions . to change the pelvic tilt angle , an adjustable angle platform to set the ankles in a particular position was used . according to the authors , the horizontal position was the neutral position of the pelvis , the dorsiflexed ankle position facilitated the anterior pelvic tilt , and the plantar - flexed ankle position caused the posterior pelvic tilt . the results of this study showed that higher pfm emg activity occurred in the horizontal position and when standing with the ankles in the dorsiflexion position ( when the pelvis tilts anteriorly ) than when standing with the ankles in the plantar flexion position . the results of chen et al35 are similar to our observations as , in the supine position ( c ) , in which the participants achieved the highest functional pfm activity , the pelvis tends to roll forward the most . in the scientific literature,3235,38,39 some authors highlight a close correlation and cross - functional connections between the pelvis , the pfm , and other structures of the area . incorrect pelvis position can contribute to topographical changes in the position of the proximal part of the urethra and bladder neck with respect to the surrounding structures , including the urogenital diaphragm.7,40 the course of the urethra in healthy women is generally straightforward , and its curvature can be observed in the strong lift of the pelvic floor , with a large filling of the rectum , or lowering of the pelvic floor and bladder . when the pelvis orientation is correct , the urethra is directed obliquely downward and forward , almost vertically , which allows the free flow of urine during relaxation of the sphincter.4145 the more perpendicular the course of the urethra with respect to the pelvic floor , the greater the closing force of its lumen is generated by the pfm . however , according to capson et al,34 in the hypolordotic posture , the angle between the urethra and the pfm is greater than in a neutral or hyperlordotic posture , which does not explain , as discussed in this study , the higher semg activity achieved in an anterior pelvic tilt position . it is believed that muscles around the hip joint ( especially , the am and gm ) may increase the activity of the pfm,4651 while the abdominal muscles , by affecting the muscle corset of the trunk , can unweight the pfm . in the current study , the most visible differences of the activity of the synergist muscles were observed within the am . the lowest values of electrical potential ( resting and functional ) of these muscles were in the supine position . however , the highest semg values were observed in position b , in which the feet were supported . a and c , the feet did not have the support , which might result in lower semg activity of the am muscles . it can also be assumed that the am would therefore be strongly activated in the standing position . a significant relationship between the activation of the am and the pfm was shown by norton and baker.52 they conducted a cough stress test in a standing position , standing with the legs crossed , and standing with the body inclined . the authors showed less urine leakage while standing with the legs crossed than while standing in a normal position ( p<0.01 ) , which may show the strong effect of the am muscles on pfm performance . furthermore , b and stien41 observed that the contraction of , eg , the adductor muscles , leads to the activation of the pfm . sapsford et al53 noticed that during slump - supported sitting , the activity of the pfm was lower compared to unsupported sitting positions ( which require greater muscle involvement ) . the authors postulated that particular abdominal exercises would activate the pfm . similar to the am , the resting and functional activities of the gm in the supine position were the lowest . the highest values of these muscles were observed in position b. morin et al54 noticed that hip muscles , mainly the rotator and gluteal group , facilitate pfm activation . although the differences in pfm activity in different positions are still controversial , a supine position seems to be the most suitable for assessing the function and mechanism of pfm , especially in women with sui.37 in light of the abovementioned scientific reports and what is reported in this article , the relationship among pfm , synergist muscles and the pelvic tilt merits further investigation . the assessment of activity of only a certain synergistic muscles ( ra , am , gm ) is a limitation of this study . in the literature , it is noted that the transverse abdominal muscle as well as external and internal oblique muscles play an important accessory functions in pfm performance . the decision on the assessment of selected muscles came out of the limitations of research equipment , which was able to evaluate simultaneously pfm and three pairs of other muscles . the new projects will take into account the results presented in this study as well as additional aspects that may also affect the increase in the bioelectrical activity of pfm . however , the evaluation of selected muscles in the tested positions in patients with pelvic floor insufficiency will be a continuation of the research conducted by the authors . the assessment of activity of only a certain synergistic muscles ( ra , am , gm ) is a limitation of this study . the attempt to evaluate the other abdominal muscles would be very meaningful . in the literature , it is noted that the transverse abdominal muscle as well as external and internal oblique muscles play an important accessory functions in pfm performance . the decision on the assessment of selected muscles came out of the limitations of research equipment , which was able to evaluate simultaneously pfm and three pairs of other muscles . the new projects will take into account the results presented in this study as well as additional aspects that may also affect the increase in the bioelectrical activity of pfm . however , the evaluation of selected muscles in the tested positions in patients with pelvic floor insufficiency will be a continuation of the research conducted by the authors . in the study , we evaluated the relationship between the activity of pfm and various lower limb positions . the results showed that in the supine position , the pfm achieved the lowest resting activity and the highest functional activity . therefore , the supine position can be recommended for the diagnosis and therapy of weakened pfm and sui . in the assessed positions , we did not observe that the synergist muscles ( am , ra , gm ) influence pfm activity . thus , the obtained pfm activity may be treated as a result measured in isolated conditions . it seems , however , that other muscles , such as the transverse abdominal muscles and abdominal oblique muscles , play a great supporting role for the pfm , which will be investigated in further studies .

a 51-yr - old female was referred to our outpatient clinic for the evaluation of generalized edema . she had been diagnosed with idiopathic thrombocytopenic purpura ( itp ) . she had taken no medicine . except for the itp , she had no history of systemic disease . she was diagnosed with systemic lupus erythematosus . immunosuppressions consisting of high - dose steroid were started . when preparing the patient for discharge , a generalized myoclonic seizure occurred at the 47th day of admission . at that time , the laboratory and neurology studies showed hyperglycemic hyperosmolar syndrome . brain mri and eeg showed brain atrophy without other lesion . the seizure stopped after the blood sugar and serum osmolarity declined below the upper normal limit . the patient became asymptomatic and she was discharged 10 weeks after admission under maintenance therapy with prednisolone , insulin glargine and nateglinide . the patient remained asymptomatic under maintenance therapy with deflazacort and without insulin or medication for blood sugar control . objectivesin physiotherapeutic practice , special attention is being given to the reciprocal anatomical , physiological , and biomechanical relationship of the pelvis and the structures connected to it . however , the scientific literature shows mainly the theoretical information about their mutual connections . the lack of information about these relations from a practical aspect coupled with the paucity of scientific papers on the impact of posture changes on the pelvic floor led the authors to conduct this study . the primary aim of this study was to compare the resting and functional bioelectrical activities of pelvic floor muscles ( pfms ) depending on three different positions of the lower limbs ( positions a , b , and c ) in the supine position.materials and methodsthis was a prospective observational study evaluating resting and functional activities of the pfm depending on the position of the lower limbs . the study was carried out at the department and clinic of urology , university hospital in wroclaw , poland and the target group were women in the menopausal period . bioelectrical activity of pfm was recorded using a surface electromyographic instrument in the supine position . results of the values obtained in a , b , and c positions were compared using a one - way analysis of variance.resultsin position a , the average resting surface electromyography ( semg ) activity of pfm was 6.92.6 v ; in position b , the result was 6.92.5 v and in position c , the resting semg activity was 5.71.8 v ( p=0.0102 ) . the results of the functional bioelectrical activity of pfm were as follows : position a 20.311.8 v , position b 19.910.6 v , and position c 25.310.9 v ( p=0.0104).conclusionthe results showed that in the supine position , the pfm achieved the lowest resting activity and the highest functional activity . therefore , the supine position can be recommended for the diagnosis and therapy of weakened pfm .

thermal spring 's microbiomes have been in attention of the scientific community since past few decades . these springs , being hub of diverse microflora , increase the probability of vast gene pool of uncultured microbiota . metagenomics have helped the microbiologist to reveal the genome of the rest of the 99% of non - cultivable microbes which further helped to better understand the global microbial ecology and also helped in meeting the current demand for novel enzymes . thus , with the advances in metagenomics all the hidden facts of the microbial ecology have been faced off . odisha being rich in bio - diversity due to seasonal / climatic variations possesses a variety of hot - springs located in different geographical locations and vary both in physio - chemical and microbial ecology parameters . mainly four major hot - springs have been reported in odisha i.e. deulajhari hot spring in angul , taptapani hot spring in ganjam , atri hot spring in khurda and tarabalo in nayagarh . deulajhari hot spring is located at about 6 km from athamallik and between 203 north latitude and 8449 east longitude . in our study , sediment sample from deulajhari hot spring ( latitude 20.74199 n , longitude 84.49206 e ) was collected from the hot spring having 69 c temperature . the v3v4 region of the 16s rrna was amplified using primers 341f , 5-cctacgggaggcagcag-3 and 518r , 5-attaccgcggctgctgg-3 with 50 ng of metagenomic dna . the amplified pcr product was purified by gel elution using minelute column ( qiagen , india ) and further leads to 150 nucleotide paired end multiplex sequencing using illumina gaiix sequencer at genotypic technology pvt . krona tool was used for plotting krona graph as depicted in fig . 1 . 2,073,312 high quality reads obtained from the hot spring with temperature of 69 c were used for further analysis . out of the total , proteobacteria ( 88.12% ) , bacteriodetes ( 10.76% ) , firmicutes ( 0.35% ) , spirochetes ( 0.18% ) , thermi ( 0.13% ) and chloroflexi ( 0.11% ) were identified at the phylum level . of the total 216 genera in this deulajhari hot spring only 53.7% were identified and 46.29% were unidentified at the genus level . since all the phylotypes , retrieved through the sequencing , do not contribute to all the taxonomic groups known till date , it indicates the significance of the diversity of deulajhari hot spring explored . an article recently published demonstrated the efficacy of eculizumab in the treatment of children with severe hematopoietic stem cell transplantation - associated thrombotic micro - angiopathy ( hsct - tma ) . we report the case of an adult with hsct - tma successfully treated with eculizumab . hsct - tma is a rare but very serious complication of allogeneic hematopoietic progenitor stem cell transplantation . several factors have been implicated in the endothelial damage which leads to hsct - tma : calcineurin inhibitors , acute graft versus host disease ( gvhd ) and cytomegalovirus ( cmv ) infection ; however , in recent years another mechanism has been described in which complement deregulation plays an important role . therefore complement - modulating therapies are beginning to gain ground in the treatment of this complication . we report the case of a 30-year - old man , diagnosed with very serious acquired bone marrow aplasia in july 2014 . he underwent progenitor stem cell transplantation of bone marrow from his hla - identical sister in july 2014 . the conditioning regimen consisted of cyclophosphamide ( 30 mg / kg / day , 7 to 4 ) , fludarabine ( 30 mg / m2/day , 5 to 2 ) and antithymocyte globulin ( 2.5 mg / kg / day , 3 to 1 ) . , the patient developed acute cutaneous and liver gvhd ( grade ii ) which initially responded to treatment with corticosteroids and etanercept . the patient was readmitted on post - transplant day 116 with diarrhea and hyperbilirubinemia ( 1.7 mg / dl , normal values 0.3 - 1.1 mg / dl ) and colonoscopy confirmed the existence of acute intestinal gvhd . after the diagnosis of acute grade iii gvhd , which was refractory to steroids , he sequentially received various lines of treatment ( corticosteroids , mesenchymal stromal cells and sirolimus ) without any response . on post - transplant day 189 , the patient developed severe bloody diarrhea ( up to 3000 ml / day ) followed by persistent rectal bleeding that required intense transfusional support and treatment with activated factor vii ( 5 mg/2 h 6 doses ) . a new colonoscopy was performed and the colonic mucosa biopsy confirmed worsening of the intestinal gvhd without histological evidence of hsct - tma ( figure 1 ) . iu / l ) , total bilirubin 0.7 mg / dl ( normal values 0.3 - 1.1 mg / dl ) , hemoglobin 8.5 g / dl , platelets 4210/l and normal coagulation tests . treatment was then initiated with one dose of pentostatin ( 4 mg / m2 iv ) and alemtuzumab ( 20 mg sc 3 times / week for 2 weeks ) . one week after the administration of pentostatin , and with persistent gastrointestinal bleeding , biochemistry showed hyperbilirubinemia ( total bilirubin 6.4 mg / dl , direct bilirubin 5.5 mg / dl , normal values 0.0 - 0.5 mg / dl ) and elevated ldh ( 2700 the blood count revealed profound anemia ( up to 6.8 g hb / dl ) , reticulocytosis ( 0.310/l ) , thrombocytopenia 3910/l and the presence of numerous schistocytes in blood smear ( 6% ) . other laboratory findings were : negative direct coombs test , undetectable haptoglobin , proteinuria ( 30 mg / dl ) , normal adamst13 activity ( 94% ) and normal complement proteins ( c3 and c4 ) . that the patient was diagnosed with hsct - tma , treatment was initiated with eculizumab 900 mg iv weekly for 4 doses followed by a single maintenance dose of 1200 mg 2 weeks later . after the first dose of eculizumab , the patient ceased to require transfusions and a progressive improvement in analytical parameters for microangiopathy was observed until their complete normalization after 7 weeks ( hb 11.4 g / dl , platelets 16410/l , no schistocytes , bilirubin 0.8 mg / dl and 450 ldh iu / l ) . ch50 determinations showed complement activity inhibition after each dose had been administered . coinciding with the improved of hsct - tma , the patient presented a clear response to his acute gvhd with disappearance of the diarrhea and bilirubin normalization ( figure 2 ) , although it has not been documented histologically . he was discharged eight weeks after the start of treatment ( post - transplant day 257 ) . unfortunately , one month later , the patient was readmitted by diarrhea ; a new colonoscopy showed intestinal gvhd relapse . the patient died two weeks after admission because of acute respiratory distress syndrome of unknown cause , with diffuse bilateral infiltrates , cardiomegaly and right pleural effusion in chest ct . plasma exchange , defibrotide , rituximab and basiliximab have been used with variable response rates . we started eculizumab instead of plasma exchange for the poor results of this treatment in hsct - tma cases associated with acute gvhd . in our case , the rapid clinical and analytical response to early treatment with eculizumab ( a humanized monoclonal antibody against complement fraction 5 ) supports the implication of the complement in its pathophysiology . the rapid response of a severe , refractory gvhd is noteworthy and suggests that the drug has a beneficial effect when used as coadjuvant therapy in this situation .

insights about the distribution of the microbial community prove to be the major goal of understanding microbial ecology which remains to be fully deciphered . hot springs being hub for the thermophilic microbiota attract the attention of the microbiologists . deulajhari hot spring cluster is located in the angul district of odisha . covered within a wooded area , deulajhari hot spring is also fed by the plant litter resulting in a relatively high amount of total organic content ( toc ) . for the first time , illumina sequencing based biodiversity analysis of microbial composition is studied through amplicon metagenome sequencing of 16s rrna targeting v3v4 region using metagenomic dna from the hot spring sediment . over 28 phyla were detected through the amplicon metagenome sequencing of which the most dominating phyla at the existing physiochemical parameters like ; temperature 69 c , ph 8.09 , electroconductivity 0.025 dsm 1 and total organic carbon 0.356% , were proteobacteria ( 88.12% ) , bacteriodetes ( 10.76% ) , firmicutes ( 0.35% ) , spirochetes ( 0.18% ) and chloroflexi ( 0.11% ) . approximately 713 species were observed at the above physiochemical parameters . the analysis of the metagenome provides the quantitative insights into microbial populations based on the sequence data in deulajhari hot spring . metagenome sequence is deposited to sra database which is available at ncbi with accession no . srx1459736 . a 30-year - old man with acquired aplastic anemia underwent an hla - identical bone marrow transplant . he developed a grade iii acute graft versus host disease ( gvhd ) refractory to various lines of treatment . on post - transplant day 196 , he was diagnosed with stem cell transplantation - associated thrombotic micro - angiopathy ( hsct - tma ) and he received treatment with eculizumab 900 mg iv weekly for 4 doses followed by a single dose of 1200 mg 2 weeks later . after the first dose of eculizumab , the patient ceased to require transfusions and a progressive improvement in analytical parameters for microangiopathy was observed until their complete normalization . coinciding with the improved of hsct - tma , the patient presented a clear response to his acute gvhd with disappearance of the diarrhea and bilirubin normalization . he was discharged eight weeks after the start of treatment . unfortunately , one month later , the patient was readmitted for a gvhd relapse and he died two weeks later by an acute respiratory distress syndrome . in our case , the rapid clinical and analytical response to early treatment with eculizumab supports the implication of the complement in hsct - tma and suggests that the drug has a beneficial effect when used as coadjuvant therapy in acute gvhd .

numerous medical conditions are known to produce psychotic symptoms that resemble schizophrenia , including metabolic and neurological disorders ( cummings 1996 ) . we describe a young woman with schizophrenia - like psychotic symptoms and functional decline in the context of diagnosed aceruloplasminemia , a disorder which has not been previously associated with psychosis . bb was a 21-year - old woman referred for neuropsychiatric assessment on the background of a psychotic illness and a confirmed diagnosis of aceruloplasminemia . bb s family had noticed an initial 18-month period of functional and social deterioration , poor school performance , social withdrawal , and periods of verbal aggression . she was initially treated by her general practitioner with fluvoxamine , but later deteriorated with frank persecutory delusions , auditory hallucinations , aggression and poor self - care over the subsequent 6 months . the local mental health team began risperidone 3 mg / day , which treated the positive symptoms but did not arrest her functional decline . bb completely avoided all social contact , only watched television and was unable to manage her activities of daily living . she was referred for neuropsychiatric assessment to further elucidate the contribution of the aceruloplasminemia to her cognitive and functional abilities . she had mild learning difficulties as a child , but attended mainstream schooling . at age 15 she had seen a school psychologist after telling other students she was going to be reborn as a prince . bb had been diagnosed with aceruloplasminemia at age 20 , after detection of abnormal liver function and hyperferritinemia of 1700 g / l ( 15200 ) . liver biopsy confirmed significant iron overload without fibrosis , and desferrioxamine 20 g thrice weekly was initiated . the diagnosis of aceruloplasminemia was highly suspected because a paternal uncle had been diagnosed with the disorder . the patient and her maternal uncle share a common , unique mutation for this disorder . bb s uncle had been described by the family as suffering long - standing behavioral problems but had never been given a psychiatric diagnosis . she was a shy and clingy child at school who had verbal and social difficulties . she left school in year 11 and attended a technical college where she also struggled . there was no history of substance abuse . on mental state examination , she was cooperative but only established superficial rapport there were no psychotic or depressive symptoms and no insight into her previous symptoms or functional impairment . on bedside cognitive testing , she scored 25/30 on the mini - mental state examination ( folstein et al 1975 ) , and 75/100 on the neuropsychiatry unit cognitive screening tool ( nucog ) ( walterfang et al 2006 ) , each significantly below expected age norms . hence , the patient was referred for a neuropsychological review , and information from an earlier assessment was obtained as a baseline . g / l ( 115150 ) with normal platelet and white cell counts and b12 and folate levels . magnetic resonance imaging ( mri ) showed global atrophy , most marked over the frontal lobes . she had a greater - than - expected load of periventricular white matter hyperintensities , and a thin anterior callosum . iron deposition was evident in the dentate nucleus of the cerebellum and the thalamus , and to a lesser degree throughout the whole cerebral cortex , but not in the basal ganglia ( figure 1 ) . a screen for lysosomal and other storage disorders on peripheral blood lymphocytes and cultured fibroblasts was normal . neuropsychological assessment showed a woman of low - average to average premorbid intellect , but demonstrating borderline general intellectual ability suggesting a significant decline . she performed significantly better on non - verbal than verbal tasks , which were poor , in addition to poor verbal memory . working memory was very poor , and psychomotor speed and speed of information processing was slowed . she displayed good performances on frontal - based tasks tapping into generating new strategies , response monitoring , inhibition , planning and organization , and mental set - shifting , although she was at times significantly slower than her age - peers . these deficits were also borne out in an occupational therapy assessment , where her deficits of new learning were manifest in requiring frequent repetition to learn new tasks , and slow mental information processing impaired her ability to deal with everyday financial and logistical tasks . the patient s risperidone was increased to 5 mg per day , with some improvement in her degree of negative symptoms without re - emergence of positive symptoms . ceruloplasmin ( cp ) is a 132-kda glycoprotein that is predominantly synthesized in the liver but does not cross the blood brain barrier and is produced by astrocytes in the cns ( klomp and gitlin 1996 ) . copper atoms incorporated during the synthesis of cp allow it to shuttle electrons and act as a multicopper ferro - oxidase . in iron homeostasis cp oxidises fe ( ferrous iron ) to fe ( ferric iron ) so that the latter can bind to transferrin and be trafficked throughout the body . fe is required as a cofactor for many biological processes but is capable of generating toxic hydroxyl and superoxide free radicals , and hence must be tightly bound to proteins during transport and storage ( de silva et al 1996 ) . cp promotes the loading of iron onto transferrin , allowing fe efflux out of cells and preventing oxidative damage caused by fe ( qian and ke 2001 ; hellman and gitlin 2002 ) . in aceruloplasminemia , an autosomal recessive disorder occurring in 1 in 2 million non - consanguinous births ( miyajima et al 1999 ) , mutations in the cp gene result in absent ferroxidase activity of cp leading to the deposition of iron in the cns , retina , pancreatic cells , liver , spleen , and ovaries ( yoshida et al 1995 ; hellman and gitlin 2002 ) . the astrocyte - specific form of cp plays a key role in regulating iron levels in the cns and in preventing free radical injury ( patel et al 2002 ) . the major sites of cns iron deposition in aceruloplasminemia are the basal ganglia , cerebellar dentate nuclei , red nucleus , thalamus , and hippocampus ( miyajima 2003 ) . the pattern of deposition , which is greatest in the globus pallidus and putamen ( miyajima 2003 ) , occurs in regions where vulnerable perivascular astrocyte populations are distributed ( klomp and gitlin 1996 ) . the sites of greatest iron concentration in healthy individuals are the basal ganglia , red nucleus , and deep nuclei of the cerebellum ( yoshida et al . aceruloplasminemia presents with retinal degeneration , diabetes mellitus and neurological symptoms , usually extrapyramidal signs , dyskinesia , dystonia , and a subcortical dementia in the fifth or sixth decade ( nittis and gitlin 2002 ) . neurological signs may be preceded for many years by diabetes mellitus and anemia due to inefficient iron delivery . mri findings typically show marked t2 hypointensity in these regions of maximal iron deposition ( morita et al . 2005 ) , although may also show subtle posterior white matter tract hyperintensity and subtle superficial cerebral and cerebellar cortical hypointensity ( grisoli et al . 2005 ) . white matter tract changes may reflect a disconnection of projecting fibres from key relay nuclei such as the thalamic nuclei , with subsequent subtle cortical changes ( grisoli et al . 2005 ) . while the clinical presentation was predominantly one of schizophrenia with no classical neurological features of aceruloplasminemia , the imaging findings of thalamic and cerebellar involvement suggest that even at this early stage cns manifestations of aceruloplasminemia are present but neurologically silent . bb s imaging also revealed prominent frontal cortical atrophy , white matter hyperintensities and thinning of the corpus callosum which are not characteristic of cp ( miyajima 2003 ) and no other pathological cause was found for these changes . regional cortical and callosal atrophy of this nature , as opposed to subtle cortical and white matter intensity changes , have not previously been reported in aceruloplasminemia . the mri findings of significant iron deposition in the thalamus and the dentate nucleus of cerebellum may be relevant to her schizophrenia - like presentation . the repeated demonstration of subtle abnormalities in structure and function of the cerebellum in schizophrenia populations has also led to its implication in the development of schizophrenia , possibly due to disruptions to the cerebellothalamo - cortico - cerebellar loop ( andreasen et al . pathology in the thalamus may also disrupt this circuit , and given that the thalamus has similarly been implicated in the pathogenesis of schizophrenia ( clinton and meador - woodruff 2004 ) , it is feasible that iron deposition - related pathology in these regions may be , in a vulnerable brain , psychotogenic . the development of psychosis in bb , atypical for aceruloplasminemia , could be seen to represent an the effects of aceruloplasminemia through pathology in dentate and thalamic regions in bb may have interacted with an early frontal insult , such as perinatal hypoxia or other obstetric complications known risk factors for the later development of schizophrenia ( cannon et al . 2002a , 2002b ) or with other unknown biological vulnerabilities , to result in schizophrenia - like symptoms during the typical age of onset for the disorder . there is a strong relationship between anatomic abnormalities in the hippocampus , cerebellum and thalamus and schizophrenia ( harrison 1999 ; shenton et al . 2001 ) and iron deposition in these regions during adolescence may have triggered a vulnerability to schizophrenia - like psychosis in this patient . we screened a collection of sponge- and marine fungi - derived extracts ( n = 2500 ) and pure compounds ( n = 400 ) using a primary cell - based in vitro model of hiv-1 latency that is re - engineered for optimal sensitivity and miniaturized for high - throughput screening . two hits were initially given high priority for further investigation : ( 1 ) the extract of a sponge putatively identified as pseudoceratina purpurea ( ucsc coll . 06135 ) , based on morphological similarity to previously identified sponges , and ( 2 ) the extract of a marine - derived fungus that was not taxonomically identified ( ucsc strain no . briefly , the known compounds psammaplin a and apicidin , shown in figure 1 , were isolated from the sponge and fungal extracts , respectively , and retested to show significant potencies ( ec50 = 0.2 m and ec50 = 0.3 m , respectively ) . a critical first step in these outcomes was the application of our recently published method for natural product peak library fractionation , whereby hplc coupled with ms and evaporative light scattering detection ( elsd ) maps the distribution of metabolites into biological screening wells as a function of retention time ( see experimental section and figure s1 , supporting information ) . subsequent operations involved scale - up isolation via peak libraries annotated with m / z ms data and bioassay hit information . apicidin has previously been shown to function as an hiv-1 reactivator via an hdaci mechanism . the successful workflow described above was employed to investigate the active extract of a marine - derived humicola fuscoatra ( ucsc strain no . 108111a ) , whose peak library elsd profile is shown in figure 2 , panel a. the major component ( tr = 2728 min ) showed a positive response in the latent hiv-1 reactivation assay . the mass spectrum ( esitof+ ) of this analyte displayed a chlorinated ion at m / z = 387.1:389.1 ( 3:1 intensity , figure 2 , panel b ) , which we duly assigned as the sodiated molecular ion [ m + na ] in conjunction with an ancillary [ m + h ] signal observed at m / z 365.1 . isolation of this active material ( ec50 = 9.1 m ) was accomplished by retrieving a pure sample from an archived library plate , and an accurate mass was measured ( esitof+ ) as 387.0637 ( [ m + na ] ) , consistent with the molecular formula of c18h17clo6 , un = 10 . conducting a literature search based upon taxonomy and this molecular formula yielded radicicol ( 1 ) , a well - known fungal metabolite and potent in vitro hsp90 inhibitor , as a solitary dereplication hit . indeed , the h nmr spectrum of the active extract ( figure 2 , panel c ) was dominated by the resonances of this metabolite . additionally , the specific rotation of + 203 for our sample from h. fuscoatra matched the literature value for 1 ( + 216 ) ; therefore , we assigned the absolute configuration of the three asymmetric carbon atoms in 1 as all r , based on a previously reported single - crystal xrd assignment . analytical data for the active extract of humicola fuscoatra ( ucsc strain no . annotations indicate the distribution of compounds 17 ( see experimental section for elution conditions ) . ( b ) esitof(+ ) m / z data collected during peak library fractionation . expansion of [ m + na ] ion for radicicol ( 1 ) , at tr = 27 min ( see panel a ) . ( c ) 600 mhz h nmr spectrum in acetone - d6 . the expanded region shows the aromatic singlet of the most abundant minor congener , compound 4 . we were inspired to further mine the extract for radicicol congeners by two pieces of information : ( 1 ) the h nmr spectrum shown in figure 2 , panel c , displayed a characteristic aromatic singlet of considerably less intensity than that of the major metabolite , and ( 2 ) the mass chromatogram generated during the peak library fractionation was rich with chlorinated ions in the region of 2026 min ( figure 2 , panel a ) . successive rounds of ms and nmr - guided hplc purification yielded minor quantities of several additional radicicol congeners : three known compounds , pochonins b ( 2 ) , c18h19clo7 , c ( 3 ) , c18h18cl2o6 , and n ( 4 ) , c18h21clo7 , and three new natural products , radicicols b d ( 57 ) . the molecular formulas and nmr properties of the known compounds radicicol b ( 5 , c18h19clo7 , un = 9 ) contains an additional molecule of h2o and one less degree of unsaturation relative to radicicol ( 1 ) . the h and c nmr data of 5 closely matched those of 1 with the following exceptions : ( i ) the h-4 and h-5 proton resonances of 1 were transposed downfield from h 3.05 and 3.32 to h 3.62 and 4.50 , respectively , and ( ii ) the c-4 and c-5 carbon resonances of 1 were transposed downfield from c 56.1 and 55.8 to c 71.9 and 69.4 , respectively . taken together with the gcosy and ghmbc correlations shown in figure 3 , the data suggest the epoxide of 1 is a 1,2-diol in 5 . the planar structure of 5 is identical to semisynthetic epoxide - opened diasteromers of natural radicicol described in both patent and peer - reviewed literature . the configuration of position c-2 is proposed to be analogous to that of radicicol ( 1 ) , and the anti relative configuration for the 1,2-diol of 5 is assigned based on a vicinal proton coupling constant of j4,5 = 5.4 hz , which matched the reported value of the synthetic anti diastereomer ( lit.j4,5 = 6.1 hz ) . the molecular formula of radicicol c ( 6 , c18h21clo8 , un = 8) contains two additional h2o molecules in comparison to 1 . the c nmr data ( table 1 ) matched those of 1 for all positions , apart from c-3 to c-10 , and suggested the two equivalents of h2o were positioned in the macrocyclic chain . with the aromatic ring functionality and macrocyclic ester linkage deemed intact , the two remaining degrees of unsaturation were assigned to resonances of a ketone ( c 204.1 ) and a trans double bond ( c 135.7 and 131.7 ; h 5.47 and 5.39 , jhh = 15.6 hz ) . additionally , three secondary alcohols were detected at h 3.60 , 3.93 , and 4.27 ( c 78.7 , 69.1 , and 70.2 ) . a single extended spin system in the gcosy spectrum was delineated from h3 - 1 to h2 - 9 , and long - range ghmbc correlations finalized the planar structure of 6 as shown in figure 3 . the stereochemistry of position c-2 is proposed to be analogous to that of radicicol ( 1 ) ; however due to a lack of both epoxide and ,,,-unsaturated ketone functionalities , radicicol c ( 6 ) is too flexible to assign the relative configurations of the three secondary alcohols by noesy correlation spectroscopy . radicicol d ( 7 ) was assigned the same molecular formula as 2 and 5 based on accurate mass measurement and required nine degrees of unsaturation . comparison of c chemical shifts to those of 1 indicated the aromatic ring functionality and macrocyclic ester linkage remained intact , and positions c-4 to c-10 were modified . two of the three remaining degrees of unsaturation were assigned to a ketone detected at c 203.7 and a cis double bond at c 129.9 and 131.6 ( h 5.98 and 6.09 , jhh = 6.2 hz ) . additionally , three oxygenated carbons were detected at c 72.3 , 84.1 , and 89.7 . the connectivity from positions 1 through 9 was established from the gcosy and ghmbc data ( figure 3 ) , and on the basis of the vicinal coupling constant of 6.2 hz between olefinic protons h-6 and h-7 , the final unit of unsaturation was assigned to a 2,5-disubstituted-2,5-dihydrofuran ring system . the stereochemistry of position c-2 in radicicol d ( 7 ) is proposed to be analogous to that of radicicol ( 1 ) ; the relative configuration of positions 4 , 5 , and 8 could not be assigned using data sets collected in this study . the possibility that radicicols b d ( 57 ) are artifacts produced during the isolation process was ruled out , as they were detected in the lcms analysis of the original etoac extract . furthermore , compounds 57 were not detected by lcms or h nmr after prolonged exposure of both natural ( 1 ) and commercial ( sigma ) radicicol samples to the isolation conditions . selected gcosy ( bold ) and ghmbc ( arrow ) correlations for radicicols b d ( 57 ) . reported values include % reactivation relative to saha and potency ( ec50 in m ) . the screening data obtained in this study are summarized in table 2 , and some of the patterns deserve additional discussion . radicicol ( 1 ) was isolated as the most abundant metabolite of h. fuscoatra and exhibited 98% reactivation efficiency of latent hiv-1 relative to the tool compound saha ( figure 4 ) and an ec50 of 9.1 m . radicicol activation of hiv-1 was confirmed by qpcr of hiv-1 rna in the same latency model ( see supplementary experimental procedures and figure s15 , supporting information ) . of the additional congeners 27 , isolated from the fungal extract , pochonins b ( 2 ) and c ( 3 ) and radicicol b ( 5 ) were reactivators ( figure 4 ) , while pochonin n ( 4 ) and radicicols c ( 6 ) and d ( 9 ) were found to be inactive . comparison of the structures of the active compounds suggests the epoxide functionality is not required for activation in our assay ; however , the active compounds all contain a michael acceptor functionality , something the inactive compounds 4 , 6 , and 7 do not possess . while the potencies in reactivating latent hiv-1 for the active radicicols and pochonins are lower than that of romidepsin , it appears that their mechanism of action is unique and remains to be elucidated . indeed , radicicol was previously reported to lack in vitro activity against histone deacetylase . although radicicol is a potent in vitro hsp90 inhibitor ( ic50 = 19 nm ) , a structurally unrelated hsp90 inhibitor , geldanamycin , is inactive in our assay , indicating that the mechanism of hiv-1 reactivation by 1 is unlikely to involve hsp90 inhibition . in addition , the most potent analogue , pochonin c ( 3 ) , is unlikely to act as an hsp90 inhibitor based on the conformational hypothesis advanced by winssinger : that hsp90 inhibition by radicicol - type resorcylides is closely tied to a specific bioactive molecular topology attenuated by modifications at the epoxide moiety . finally , radicicol did not induce detectable nfkb reporter gene activation in jurkat cells ( see supplementary experimental procedures and figure s15 , supporting information ) , indicating it reactivates latent hiv-1 by a pkc - independent mechanism . in vitro latent hiv-1 reactivation assay : dose response profiles of radicicol ( 1 ) , pochonin b ( 2 ) pochonin c ( 3 ) , and radicicol b ( 5 ) relative to the maximum activation of the positive control saha . . a screening paradigm has been created consisting of a sensitive and high - throughput primary cell - based hiv-1 latency assay that was well suited to investigate diverse marine natural product collections for novel reactivators of latent hiv-1 . the assay was robust with highly reproducible ec50 values for reference compound saha in all donors used in this screen . starting from an extract of the marine - derived fungus h. fuscoatra and followed by successive rounds of purification , dereplication , and bioassay , we isolated seven compounds in the radicicol / pochonin class , of which four display latent hiv-1 reactivation activity , providing a preliminary understanding of the structure activity relationship of these compounds . at this stage we believe that the new hiv-1 reactivators summarized in table 2 can be either the seeds for further scaffold modification or useful in combination with agents possessing a distinct mechanism of action , such as romidepsin , to achieve a higher magnitude of latent hiv-1 reactivation . standard pulse sequences were used for all nmr experiments , which were run on either a varian unity inova spectrometer ( 600 and 150 mhz for h and c , respectively ) equipped with a 5 mm triple resonance ( hcn ) cold probe or a varian spectrometer ( 500 and 125 mhz for h and c , respectively ) equipped with an inverse detection probe . residual solvent shifts for acetone - d6 were referenced to h 2.05 and c 29.84 , respectively . accurate mass measurements were obtained on a mariner esitof instrument for molecular formula determinations . all chromatographic work was done in reversed - phase and utilized hplc grade ch3cn ( solvent a ) and milli - q h2o ( solvent b ) , both adjusted to contain 0.1% formic acid . the analytical lcms system was composed of waters hplc components ( i.e. , solvent pumps and autosampler ) and controlled by empower software . a 150 4.60 mm 5 m luna c18 column ( phenomenex ) was utilized , and the system operated at a flow rate of 1 ml / min . the postcolumn flow of the eluent was first through a photodiode array ( waters ) and then split ( 1:1 ) between an evaporative light - scattering detector ( sedex model 55 ) and an esitof mass spectrometer ( applied biosystems mariner ) . the semipreparative hplc system was composed of waters hplc components ( i.e. , solvent pumps and gradient controller ) and equipped with a 250 10 mm 5 m luna c18 column ( phenomenex ) and tunable uv - absorbance detector ( waters ) . the humicola fuscoatra strain ( ucsc coll . 108111a ) was isolated from sediment collected at <33 m depth by scuba in tutuila , american samoa ( s 1416.600 , w 17036.719 ) , in 2010 . the strain was taxonomically identified by molecular ( 100% by its and d1/d2 regions of rdna ) and morphological methods at the university of texas fungus testing laboratory . the 125 ml and 5 l cultures of 108111a were grown in media that consisted of the following : cornmeal ( 4.5 g/150 ml ) and 5xasw ( 200 after autoclave sterilization , the cultures were inoculated and shaken at 75 rpm for 21 days at ambient temperature . at the time of harvest , the fungal cultures ( 5 1 l ) were homogenized , and the liquid was extracted three times with equal volumes of etoac . the combined organic extracts were concentrated in vacuo to yield 2 g of oil . a portion of the 108111a cma extract ( 194.9 mg ) was separated into 15 fractions ( fraction codes h1h15 , figure s2 ) using the semipreparative hplc system utilizing a flow rate of 3 ml / min , 310 nm detection , and the following elution conditions : 15 min isocratic 25:75 , 25 min gradient from 25:75 to 70:30 , 3 min gradient from 70:30 to 100:0 , 10 min isocratic 100:0 , and 3 min gradient from 100:0 to 25:75 . fraction h5 was pochonin n ( 4 , 5.0 mg ) , fraction h9 was pochonin b ( 2,1.8 mg ) , and fraction h9 was pochonin c ( 3 , 1.9 mg ) . fraction h12 was semipure radicicol , and an additional run under identical hplc conditions yielded pure radicicol ( 1 , 11.2 mg ) and three new analogues : radicicol b ( 5 , 1.2 mg ) , radicicol c ( 6 , 2.2 mg ) , and radicicol d ( 7 , 1.0 mg ) . the instrumentation setup for our natural product peak library generation has been recently published . the hplc stage utilized two waters 510 pumps and a waters 717plus autosampler , both controlled with empower 2 software . separation was performed on a 250 10 mm 5 m luna c18 column ( phenomenex ) . spectra from three detectors were acquired during peak library fractionation : waters 996 photo diode array , sedex 55 elsd , and mariner 5054 esi - tof - ms . the mobile phase parameters are ch3cn ( a ) and h2o ( b ) with a flow rate of 2 ml / min and the following elution conditions : 30 min gradient from 10:90 , 10 min isocratic 100:0 , 1 min gradient from 100:0 to 10:90 , 9 min isocratic 10:90 . sample collection was performed using a gilson 215 liquid handler controlled with gilson unipoint lc software . samples were collected into bd biosciences 96-deep - well plates , with a working volume of 2 ml ( part number : 353966 ) . after the lc - ms - uv - elsd library is collected , a duplicate archive plate is generated for analytical reference using a 12-channel pipet , creating an exact copy and counter balance for centrifugal drying . [ ]d + 216 ( c 1.00 , chloroform ) ; uv , h nmr , c nmr , and hresitofms data were in accordance with reported values . [ ]d + 20 ( c 1.0 , acetone ) ; uv , h nmr , c nmr , and hresitofms data were in accordance with reported values . ( )-2r,4s,5r [ ]d 68.3 ( c 0.06 , chloroform ) ; uv , h nmr , c nmr , and hresitofms data were in accordance with reported values . [ ]d 77.3 ( c 1.00 , methanol ) ; uv , h nmr , c nmr , and hresitofms data were in accordance with reported values . [ ]d + 32 ( c 0.8 , methanol ) ; uv ( lc eluent h2o / ch3cn / formic acid , 1:1:0.1 ) max ( au ) 219 ( 0.87 ) , 276 ( 0.60 ) nm ; h and c nmr data , see table 1 ; hresitofms m / z 405.0739 [ m + na ] ( calcd for c18h19clo7na , 405.0712 ) . [ ]d 69 ( c 1.0 , methanol ) ; uv ( lc eluent h2o / ch3cn / formic acid , 1:1:0.1 ) max ( au ) 219 ( 0.94 ) 262 ( 0.36 ) 309 ( 0.20 ) nm ; h and c nmr data , see table 1 ; hresitofms m / z 423.0826 [ m + na ] ( calcd for c18h21clo8na , 423.0817 ) . [ ]d 53 ( c 0.4 , methanol ) ; uv ( lc eluent h2o / ch3cn / formic acid , 1:1:0.1 ) max ( au ) 217 ( 0.48 ) 257 ( 0.12 ) 297 ( 0.08 ) nm ; h and c nmr data , see table 1 ; hresitofms m / z 405.0668 [ m + na ] ( calcd for c18h19clo7na , 405.0712 ) . an nl4 - 3-based vector with frame - shift mutations in vpr and env genes was engineered with a codon - optimized firefly luciferase gene in place of nef to generate the resulting construct pks13 . the nl4 - 3-luc virus was generated by co - transfection of hek-293 t cells with pks13 and a plasmid containing the hiv-1 env gene using lipofectamine 2000 ( life technologies ) . briefly , naive cd4 + t cells were purified by negative selection using pbmcs from healthy hiv-1 negative donors . purified naive cd4 + t cells were activated by incubation with anti - cd3/cd28 magnetic dynabeads ( 1 bead:2 cells ratio , life technologies ) , 1 mg / ml anti - il-4 ( r&d systems ) , 2 mg / ml anti - il-12p70 antibodies ( r&d systems ) , and 10 ng / ml tgf- ( r&d systems ) for 3 days . cells were maintained in 30 u / ml il-2 ( life technologies ) for 2 days followed by infection with nl4 - 3-luc . seven days postinfection , 20 l of latently infected cells at 10 000 cells / well was dispensed into 384-well plates containing 350 nl of compound solutions in 50 l of culture medium . after a 48 h incubation , 40 l / well briteglo ( promega ) was added , and luminescence measured using the envision plate reader ( perkin - elmer ) .

schizophrenia - like illnesses occur in a variety of medical and neurological conditions but to date have not been described in association with aceruloplasminemia . aceruloplasminemia is an autosomal recessive disorder of iron metabolism which leads to iron deposition in the basal ganglia , thalamus , cerebellum and hippocampus and which usually presents in middle age with extrapyramidal symptoms and dementia . we describe a 21-year - old woman on treatment for aceruloplasminemia who presented with schizophrenia - like psychosis and declining function in the absence of neurological signs . neuropsychological testing showed significant dominant hemisphere deficits . magnetic resonance imaging showed bilateral iron deposition in the cerebellar dentate nuclei and thalami , frontal atrophy , and periventricular white matter hyperintensities . functional imaging suggested global hypoperfusion . the clinical , cognitive and imaging findings were not typical for either aceruloplasminemia or schizophrenia alone and the possible relationship between the two disorders is discussed with particular reference to implications for our understanding of schizophrenia . an extract of humicola fuscoatra ( ucsc strain no . 108111a ) was shown to reactivate latent hiv-1 expression in an in vitro model of central memory cd4 + t cells . we report the bioassay - guided isolation and structure determination of several resorcyclic acid lactones , including four known compounds , radicicol ( 1 , aka . monorden ) and pochonins b ( 2 ) , c ( 3 ) , and n ( 4 ) , and three new analogues , radicicols b d ( 57 ) . compounds 13 and 5 showed moderate activities in the memory t cell model of hiv-1 latency . radicicol ( 1 ) displayed lower potency in reactivating latent hiv-1 ( ec50 = 9.1 m ) relative to the hdac inhibitors apicidin ( ec50 = 0.3 m ) , romidepsin ( ec50 = 0.003 m ) , and saha ( ec50 = 0.6 m ) ; however , it achieved equivalent maximum efficacy relative to the positive control compounds ( 98% of saha and romidepsin ) .

warfarin is highly effective in reducing stroke risk in patients with atrial fibrillation ( af ) . however , using warfarin is not easy ; effort is needed to educate patients about drug and dietary interactions , and serial monitoring of the international normalization ratio ( inr ) is required . as a result , a recent population - based cohort study showed that 43% of patients stopped warfarin within 3 years and 61% within 5 years.1 in addition , warfarin has a narrow therapeutic threshold , and the quality of anticoagulation has a critical influence on its effect . in a uk cohort study , warfarin therapy with a time in therapeutic range ( ttr ) of inr of less than 60% did not significantly reduce stroke risk compared to no antithrombotic therapy.2 in a post hoc analysis of active w ( atrial fibrillation clopidogrel trial with irbesartan for prevention of vascular events ) , in order to ensure the superiority of warfarin over clopidogrel plus aspirin the minimum projected ttr threshold was 58%.3 while suboptimal doses lead to an increased risk of ischemic stroke , overdosing frequently results in hemorrhage that can be fatal . even in the setting of clinical trials , the reported ttrs were only about two thirds.4,5,6,7 it also has been shown that eight adverse bleeding events are estimated to occur annually for every 100 patients treated,8 and the underutilization of warfarin is in part due to concerns about hemorrhagic risk . lastly , the use of warfarin is particularly cumbersome in old subjects and in those with stroke . they are more vulnerable to bleeding complications that may be related with poor compliance , cognitive impairment , and the frequent presence of comorbid diseases that require polypharmacy and invasive procedures . in subjects undergoing invasive treatment , periprocedural management is difficult and carries a high risk for thromboembolism , partly because of the slow onset and offset of action ( long half - life ) of warfarin . therefore , more ideal anticoagulants are required that have a wide therapeutic range , a short half - life , and a predictable therapeutic effect with fixed or weight - based dosing , without significant food or drug interaction.9 several warfarin alternatives have been tested but failed to show benefits because of serious adverse effects , i.e. , excessive bleeding with idraparinux10 or hepatic dysfunction with ximelagatran.11 new oral anticoagulants ( noacs ) ( dabigatran , apixaban , rivaroxaban , and edoxaban ) were first introduced in 2005 , for prophylaxis against venous thromboembolism after total hip or knee replacement surgery , and are now used to prevent and treat deep vein thrombosis and pulmonary embolism ( table 1).12 more recently , four large phase iii randomized controlled trials of noacs ( re - ly , rocket - af , aristotle , and engage - af - timi 48 ) have been completed in patients with non - valvular af.4,5,6,7 overall , noacs were superior or non - inferior to warfarin in the prevention of ischemic stroke and systemic embolism , but superior to warfarin in reducing hemorrhagic stroke.4,5,6,7 noacs may be more beneficial among patients with af - related stroke or transient ischemic attack ( tia ) ; a recent meta - analysis showed that noacs significantly reduced stroke or systemic embolism , as well as hemorrhagic stroke or major bleeding , in patients with af and previous stroke or tia.13 the relative risk reduction and the number needed to treat with noacs vs. warfarin to reduce stroke / systemic embolism were estimated to be 14% and 134 patients ; for avoiding hemorrhagic stroke the respective values were 57.9% and 139 patients.13 evidence from these trials forms the basis for national and international guidelines for the management of patients with non - valvular af in clinical practice . although aspirin was preferred in patients with a low - risk of stroke , new and safer anticoagulants may lead to lowering of the threshold for anticoagulation.14 the selection of antithrombotic agent ( warfarin vs. noacs ) should be individualized on the basis of risk factors , cost , tolerability , patient preference , potential for drug interactions , and other clinical characteristics such as renal function and ttr.15 it would be unnecessary to switch from warfarin to noacs in patients who already achieve stable and good inr control with warfarin . good anticoagulation control ( ttr>70% ) is associated with the best efficacy and safety of warfarin,2 and the recent meta - analysis of the four phase iii noacs trials showed that there was a greater relative reduction in major bleeding with noacs when the ttr was less than 66% than when it was greater.16 first of all , it appears that asians more often have cerebral small vessel disease , which is prone to bleeding . it has been shown that hemorrhagic stroke ( intracerebral or subarachnoid hemorrhage ) , which accounts for 15% to 20% of strokes in whites,17 is more frequent among asians.18 death from hemorrhagic stroke is also much more common in asians.19 the prevalence of cerebral microbleeds , which increase the risk of intracerebral hemorrhage on anticoagulation , also appears to be higher in asian than in white population.20 the ethnic differences in bleeding - prone small artery disease are in part related with the high prevalence of uncontrolled hypertension or hypocholesterolemia in this region.21 ethnic differences in the salt sensitivity of intracranial hemorrhage ( ich ) have also been addressed.22 whatever the reason , these small artery diseases predispose patients to cerebral bleeding when they are given antithrombotics ; the presence of cerebral small artery diseases , such as lacunar infarcts , white matter ischemia or microbleeds , increases the risk of ich in patients receiving antiplatelet agents23 or anticoagulants.24,25 a recent study showed a high prevalence of warfarin - associated ich in asians , even if the inr levels were comparable ; the hazard ratio for ich was 4.06 for asians , and 2 for hispanics and blacks compared to whites.26 considering that noacs have a lower bleeding risk than warfarin , the benefit of noacs may be greater in asian patients . second , asians generally favor complementary treatment , such as herbal medication and healthy food . according to a study from korea , 40% of patients who were initially treated in a large tertiary hospital tried additional herbal treatment after discharge.27 this is in strong contrast to a report from canada , where patients with cerebrovascular diseases rarely used alternative medicine.28 a study from hong kong found that 26% of patients taking warfarin had ingested herbal remedies.29 this behavior probably results in significant warfarin - drug / food interaction , poor inr control , and may at least in part explain the lower ttr achievement in asian ( mean 54.5% ) than in non - asian ( 66.2% ) patients in the re - ly trial.30,31 as asians more often had a lower ( rather than higher ) inr level , another explanation would be that asian physicians tend to target a relatively low inr because of the fear of bleeding complications . indeed , japanese guidelines recommend a target inr of 1.6 - 2.6 for japanese people.32 whatever the reason , inr control is relatively poor in asia , which also supports the notion that noacs may be more suitable for asians . finally , there has been evidence that population frequencies of genetic polymorphisms related with warfarin metabolism and action ( e.g. , cyp2c9 and vkorc1 ) differ according to ethnicities.33,34,35 apart from being linked to ethnic variations in the required warfarin dose , these differences may also partly explain the poor inr control and the higher risk of ich among asians . recent studies report that noacs may be particularly valuable in asians compared to non - asians.31,36,37 the re - ly investigators reported that , among patients treated with warfarin , asians ( vs. non - asians ) had higher rates of ischemic stroke ( by 2.1-fold ) as well as hemorrhagic stroke ( by 2.4-fold ) , despite similar blood pressure , younger age , and relatively lower inr values . the magnitude of the benefit from dabigatran in the prevention of ischemic stroke and hemorrhagic was greater in asians than in non - asians.31 more recently , chiang and colleagues reported a post - hoc analysis of the asian population included in the re - ly and arisotle trials , which showed that , compared to non - asians , asians had a lower number needed to treat with noacs vs. warfarin to reduce stroke / systemic embolism , and a higher number needed to treat to avoid hemorrhagic complications , favoring their use in asians in terms of both efficacy and safety.38 cost - effectiveness analyses showed that noacs are cost - effective relative to warfarin for stroke prevention in patients with af and stroke / tia.39,40,41 cost - effectiveness is associated with the cost of the drug , care costs , the loss of productivity due to disability following recurrent disease , individual patient risk for thromboembolism and bleeding , and the quality of anticoagulation control ( i.e. , ttr ) which in turn is related to compliance , warfarin pharmacogenomics , and accessibility for regular inr tests . we suggest that noacs may be particularly cost - effective in asian stroke patients for the following reasons . first , the risk of both recurrent thromboembolism and bleeding is higher in stroke patients than in patients without stroke . moreover , as discussed above , asians are at higher risk of warfarin - related ich , a devastating condition , than caucasians . second , patients with af - related stroke have a poorer control of inr than those without stroke , probably because of more severe neurological disability and a lack of access to frequent inr monitoring . it has been shown that most ( > 70% ) af - related stroke patients have lobar or territorial infarcts,42 and poor anticoagulation control is associated with the size of brain infarcts ( figure 1).43 thus , asian stroke patients more often face problems in using warfarin : difficulty in maintaining inr and frequent bleeding complications . in this . however , noacs are expensive drugs and studies focusing on the cost - effectiveness of noacs in asian stroke patients are unavailable . nevertheless , given the considerations so far discussed , we propose that the reimbursement policy for the use of noacs should be more generous for asian stroke patients . the guidelines for the reimbursement of noacs are summarized in table 2 . in japan , taiwan , norway , switzerland , iceland , and the united arab emirates , there is no restriction as long as noacs are prescribed on - label . the reimbursement guidelines for noacs in some european countries , including the uk , france , and germany , are based only on the chads2 score ( 1 ) . other european countries and canada include both chads2 score and the quality of anticoagulation control . for example , the provincial reimbursement guideline for noacs in canada includes ( a ) unable to tolerate warfarin , ( b ) inrs not well controlled on warfarin , or ( c ) unable to have inrs tested regularly , in patients with a chads2 score of 2 . the guidelines for the reimbursement of noacs in korea are similar to those of canada , but the accessibility ( ability to have inr tested regularly ) is not considered . the criteria for ' inadequate anticoagulation ' appear to be too strict , especially during the maintenance period ( table 3 ) . as a result , it is difficult to prescribe noacs to stroke patients with severe disability , or those who live in remote areas ( e.g. , on an island ) and are thus unable to test inr regularly . the guideline also recommends a frequent change of warfarin dose ( at least 5 times during 6 months ) to monitor inr . this is in conflict with a randomized study comparing ttr between warfarin dose assessment every 4 weeks vs. every 12 weeks , which showed that frequent dose changes to monitor inr are unnecessary and result in temporary fluctuation of the inr.44 moreover , the guidelines state that reimbursement can be allowed if patients have a major bleeding complication during warfarin treatment . from the strokologist 's point of view this does not make sense , because warfarin - associated ich is frequently fatal . we understand that the strict guidelines can be attributed to the high cost of noacs , which may adversely affects the health budget , but we would suggest that the guidelines be more generous to asian stroke patients , who are particularly vulnerable to the adverse effects of warfarin . we suggest that this approach may be more cost - effective than not using noacs , because recurrent ischemic stroke or ich will be very costly in this rapidly aging society . as discussed above , cost - effectiveness studies on asian stroke patients are unavailable and should be performed in the future to answer this question . af is an emerging global epidemic in both high - income and low - income countries . recently , the use of noacs in af patients has been rapidly increasing , but the cost of noacs is unaffordable in low - income to middle - income countries . . however , the cost - effectiveness of noacs will depend on the characteristics of patients as well as therapy pricing . asian countries are facing population aging at a pace that is unprecedented and an increasing burden of stroke , particularly af - related stroke . af - related stroke is more disabling and more fatal than other stroke subtypes , and is expected to increase substantially in the decades to come ( figure 2 ) . compared to caucasians , asians more often develop hemorrhagic stroke and warfarin - related ich , as well as any stroke in the case of patients with af , and the benefit with noacs vs. warfarin for preventing stroke and reducing anticoagulation - related ich would be particularly high in asians . nevertheless , the current reimbursement guidelines are in general suboptimal . we suggest that guidelines for using noacs should be more generous for asian stroke patients . in addition , as noacs are expensive , continuous efforts should be made by healthcare providers and governors to find the ideal candidate for noacs ( most cost - effective group ) ; this includes research to find genetic or clinical factors that predict the quality of anticoagulation control.45,46 close communication is needed to arrive at a consensus decision regarding the coverage guideline for those patients who need noacs , and to reduce the ever - increasing burden of af - related stroke . the author has not received any funding or benefits from industry or elsewhere to conduct this study .

atrial fibrillation ( af ) is an emerging epidemic in both high - income and low - income countries , mainly because of global population aging . stroke is a major complication of af , and af - related ischemic stroke is more disabling and more fatal than other types of ischemic stroke . however , because of concerns about bleeding complications , particularly intracranial hemorrhage , and the limitations of a narrow therapeutic window , warfarin is underused . four large phase iii randomized controlled trials in patients with non - valvular af ( re - ly , rocket - af , aristotle , and engage - af - timi 48 ) demonstrated that new oral anticoagulants ( noacs ) are superior or non - inferior to warfarin as regards their efficacy in preventing ischemic stroke and systemic embolism , and superior to warfarin in terms of intracranial hemorrhage . among af patients receiving warfarin , asians compared to non - asians are at higher risk of stroke or systemic embolism and are also more prone to develop major bleeding complications , including intracranial hemorrhage . the extra benefit offered by noacs over warfarin appears to be greater in asians than in non - asians . in addition , asians are less compliant , partly because of the frequent use of herbal remedies . therefore , noacs compared to warfarin may be safer and more useful in asians than in non - asians , especially in stroke patients . although the use of noacs in af patients is rapidly increasing , guidelines for the insurance reimbursement of noacs have not been resolved , partly because of insufficient understanding of the benefit of noacs and partly because of cost concerns . the cost - effectiveness of noacs has been well demonstrated in the healthcare settings of developed countries , and its magnitude would vary depending on population characteristics as well as treatment cost . therefore , academic societies and regulatory authorities should work together to formulate a scientific healthcare policy that will effectively reduce the burden of af - related stroke in this rapidly aging society . there remain tremendous opportunities to improve the stability and safety of american health care . within this context , residents and residency programs face two essential questions : how to reduce the risk to patients resulting from resident inexperience , and how to change our programs to create the safer physician of the future ? the spread of side - by - side teaching and non - teaching services creates a natural setting to study these questions and improve both services . when asked the question , would you admit your mother to the resident service ? , many of us respond , it depends . we are focusing this column on helping programs answer this question definitively in the positive , share potential best practices , and underscore community hospital 's contribution to our understanding of patient safety .

attention to women 's health has evolved from a limited focus on reproductive and breast health to encompassing all aspects of health from birth through old age . in the era of individualized and precision medicine , innovative medical , surgical , pharmacological , regenerative , and genetic technologies are available to improve the health of women . application of these technologies increases the potential to better diagnose and treat conditions , both acute and chronic , that are specific to women , occur more frequently in women than men , or that present with different symptoms and outcomes for women compared to men . to address the evolving definition of women 's health , health care and academic research institutions have established women 's clinical and research centers that encompass holistic approaches to women 's health and include gynecological , reproductive , and obstetric services , as well as other medical subspecialties ( e.g. , cardiology , gastroenterology , immunology , hematology , endocrinology , nephrology , neurology , rehabilitation medicine , nutrition , and psychiatric services ) . many of these centers were developed as a result of government - funded initiatives promoted by the office on women 's health in the department of health and human services ( i.e. , the centers of excellence in women 's health ) and by the office of research on women 's health ( orwh ) at the national institutes of health ( i.e. , the specialized centers of research on sex differences ) , and the training program building interdisciplinary research careers in women 's health ( bircwh ) . critical to the future advances in women 's health is continued research into all aspects of female physiology and pathophysiology to provide the evidence base for practice guidelines and to educate the next generation of basic science investigators , clinical investigators , and healthcare providers . the national institutes of health 's ( nih ) commitment to improving health outcomes for women and men through rigorous science has been compromised by the lack of basic science evidence obtained from females animals . to correct this limitation , in june 2015 the nih announced expectations that sex , as a biological variable , be included into research design and analysis in studies of vertebrate animals and humans ( not - od-15 - 102 ) . however , caution is needed in how this directive is interpreted by scientists and study sections who review grant applications . there is a risk that their interpretation may restrict attention to studies involving the direct comparison between female / women and male / men ignoring the contribution of sex - specific conditions . understanding how sex influences all aspects of health and disease needs to take a programmatic approach that includes the study of sex - specific conditions . legislation reintroduced into the united states house of representatives in the form the research for all act 2101 ) codifies the nih support for the specialized centers of research on sex differences . first , the language of the final bill should not be restrictive , but rather flexible so that the centers can expand their research scope to follow scientific discoveries . second , in the need to increase understanding of the physiological processes that differ between women / females and men / males , it is possible to ignore those processes related to sex - specific conditions ( i.e. , those related to reproduction ) . the exclusion of the study of sex - specific conditions may actually slow progress in understanding women 's health across the life - span . said another way , because there are sex - specific conditions , there are sex differences in all physiological processes . women 's health , viewed through the lens of sex differences , incorporates research into reproductive health and the physiological processes directed by the xx chromosomal complement , including hormonal changes accompanying puberty , pregnancy , and menopause that have lifelong consequences . genomic analysis is a strategic part of the national precision medicine initiative ( www.nih.gov/precisionmedicine ) . implied but not stated in the publically available web material however , inclusion of the sex chromosomes in genome - wide association studies ( gwas ) analysis to date is sparse , and we hope this new precision medicine initiative will increase attention to the contribution of the sex chromosomes to health and disease . investigators in women 's health research are developing statistical packages that consider the sex chromosomes and x chromosome inactivation in gwas . including analysis of the x chromosome is critical to understand sex differences and to ensure the success of the national precision medicine initiative . the future of women 's health requires development of models to sustain basic and clinical research and educational initiatives in women 's health in the absence of , or in spite of , specific government directives . women 's health must become a natural part of the culture of clinical practice , discovery science , and medical / health education . mayo clinic has embraced individualized ( precision ) medicine as a strategic investment in the future of health care . the strategy is an evidence - based practice built on basic discoveries of the root causes of disease . by definition , a cornerstone of precision care is to recognize the contribution of biological sex ( as dictated by the complement of sex chromosomes ) and of gender ( that includes cultural and environmental influences that define feminine and masculine ) . some women 's health clinics and research centers focus on specific patient subsets and activities that reflect the clinical and research expertise of their faculties . the specialized centers of research on sex differences funded by the nih in association with orwh represent a model for the development of interdisciplinary teams . these centers act as catalysts to discovery and advancement of knowledge to improve the quality of care for women by incorporating both sex differences and sex - specific conditions . this approach also can be applied more broadly in other research and clinical areas not typically considered for example , ongoing research in women 's health and sex - based medicine at mayo clinic crosses multiple specialties . these programs include collaborations between engineers , primary care providers , and oncologists to develop imaging modalities that can better detect breast cancers in women with dense breasts that are not easily identified by conventional mammography . gynecological surgeons partner with oncologists and molecular scientists to develop non - invasive and sensitive tests to detect endometrial cancer . epidemiologists partner with gynecologists and internists to identify health disparities related to uterine fibroids in ethnic minorities , risks of overall mortality , and chronic diseases in women who have undergone oophorectomy , and conditions of pregnancy such as hypertension , preeclampsia , eclampsia , and gestational diabetes that affect lifelong risk for cardiovascular disease in women . however , in addition to these more typical women 's health arenas , cardiologists partner with basic scientists and surgeons to investigate sex differences in the etiology of valvular calcification and heart disease , and with neurologists to evaluate how the autonomic nervous system contributes to the etiology of postural orthostatic hypotension and fibromyalgia . with the aging of the population , investigation of sex - specific molecular pathways associated with age - related diseases such as sarcopenia are being pursued to understand how interventions may slow these processes . radiologists are working with neurologists to develop improved methods to detect sex - specific alterations in brain structures associated with cognitive decline . these initiatives are probably not unique to our organization but most likely reflect similar activities at other academic medical centers . these projects share an understanding that there are sex differences in the physiological processes under investigation and that these differences impact women 's health . thus , sex and hormonal status are key biological variables for organ and tissue transplantation , regenerative medicine , and pharmacogenomic programs . the support for sex - specific as well as sex - different initiatives will require dedicated medical services and patient - specific biorepositories . for example , sex - specific clinical databases can be developed within existing women 's health clinics such as the data registry on experiences of aging , menopause , and sexuality ( dreams ) developed by the mayo clinic women 's health clinic . the dreams project was designed to study , for example , the effects of caffeine intake on menopausal symptoms , women 's views of menopause and the symptoms experienced , and the association of recent physical and mental abuse with menopausal symptoms . similar clinical initiatives provide resources to junior investigators in women 's health to develop longitudinal studies of women as they age . discoveries from research related to women 's health and concepts of sex and gender differences in physiology and pathophysiology must become embedded into the material used to train future scientists and health care providers . programs such as the bircwh need to be sustained and expanded to create a critical mass of investigators leading and catalyzing research teams in women 's health . community , academic , national , and global partnerships are developing innovative educational materials and curricula to train the next generation of women 's health scientists and healthcare providers . opportunities are available to develop and test these curricular materials focusing on sex- and gender - based evidence in all phases of health care education through projects supported in part by the nih , the orwh , the american medical women 's association , the canadian institutes of health research , the european gender medicine program , the society for women 's health research , the organization for the study of sex differences , and the sex and gender women 's health collaborative . utilization of these educational materials will stimulate future research , facilitate translation of discoveries into patient care , and ultimately will reduce health disparities for women by improving their care . the patient through a sex and gender lens is a first step toward personalizing care . however , personalized healthcare must be based on evidence derived from research designed to study how sex and hormonal status influence health across the life - span . the mayo clinic approach to translation of women 's health research into clinical practice is embodied by innovative technologies driving discovery in regenerative medicine , organ and tissue transplantation , and pharmacogenomics . this approach may serve as a model by which other academic institutions can expand their women 's health research programs . ongoing initiatives in healthcare education are required to assure that future healthcare providers , researchers , and educators recognize that sex matters when making decisions about prevention , diagnosis , disease management , and patient outcomes . a plan to successfully integrate sex difference research and thymic neuroendocrine ( ne ) tumors associated with multiple endocrine neoplasia type 1 ( men-1 ) are rare , variably documented in 1 - 8% cases . thymic ne tumors are usually detected about 7 - 29 years following surgical treatment of primary hyperparathyroidism ( phpt ) in men-1 . transcervical thymectomy ( tct ) at the time of parathyroid surgery for phpt usually prevents thymic ne tumors . the occurrence of thymic ne tumors is very rare after tct as part of the parathyroidectomy procedure for phpt . here we report a case of thymic ne carcinoma developing within a span of 8 months after subtotal parathyroidectomy and tct for phpt in a men-1 patient . a 56-year - old non - smoker male presented with generalized bodyaches , low back pain , and proximal myopathy of 1-year duration without any fragility fractures . examination revealed bony tenderness over the sternum and proximal muscle weakness of the lower limbs . laboratory evaluation revealed hypercalcemia ( serum total calcium 12.8 mg / dl , normal 8.5 - 10.5 mg / dl ) , hypophosphatemia ( serum phosphorus 2.1 mg / dl , normal 2.5 - 4.5 mg / dl ) , normal serum albumin ( 3.8 g / dl ) , normal serum alkaline phosphatase ( 136 iu / l , normal 50 - 150 iu / l ) , vitamin d sufficiency ( serum 25oh vitamin d 31 ng / ml ) , and normal renal function ( serum creatinine 1.2 mg / dl ) . hypercalcemia was pth dependent ( serum calcium 12.8 mg / dl with high serum intact pth 215 pg / ml , normal 15 - 70 pg / ml ) . his hemogram and esr were unremarkable ( hb 13.4 g / dl , tlc 6700/mm , platelet count 2.8 lakhs / mm , and peripheral smear showed normocytic , normochromic rbcs , esr 20 mm at end of the first hour ) . bone mineral density by dual energy x - ray absorptiometry showed osteoporosis ( t - score 2.7 at lumbar spine , 2.0 at total hip , and 3.4 at distal forearm ) . ultrasound neck revealed multiglandular parathyroid enlargement , while a tc tetrofosmin parathyroid scan showed a right inferior parathyroid tumor . the patient was advised surgery as he had serum calcium > 12 mg / dl and osteoporosis . the patient was subjected to bilateral neck exploration , where asymmetrical parathyroid hyperplasia was found . histopathology showed multiglandular parathyroid hyperplasia and a normal thymus . on follow - up , 8 months after parathyroidectomy , he presented with heaviness in the left side of the chest , dry cough , and exertional dyspnea of 3 months duration . chest radiograph revealed mediastinal widening with an oval well - circumscribed homogenous opacity ( figure 1b - dark arrow ) in left hemi - thorax . contrast - enhanced computed tomography ( ct ) of thorax showed a 10 6.7 cm inhomogenously enhancing anterior mediastinal soft tissue mass in the prevascular space on the left side , in close proximity to the ascending aorta and arch of aorta [ figure 2 ] . chest radiograph done 9 months earlier [ figure 1a ] did not show any mediastinal widening . chest radiographs done 9 months apart radio - opacity in the left hemi - thorax in july 2009 radiograph ( marked with arrow ) was not present in september 2008 radiograph contrast - enhanced computed tomogram of the thorax showing large solid anterior mediastinal mass ( marked with arrow ) to the left of mid - line tc methylene diphosphonate ( mdp ) whole - body skeletal scan showed increased tracer uptake at thoraco - lumbar vertebrae and multiple ribs suggestive of skeletal metastases . the anterior mediastinal mass was excised in toto via trans - sternal approach . on surgery , the tumor was not found to invade any of the adjacent structures . the excised tumor measured 12 7 5 cm and weighed 264 g [ figure 3 ] . on microscopy [ figure 4a and b ] , the tumor was partially encapsulated , and was composed of rosettes of tumor cells traversed by thin fibro - vascular septae . the tumor cells displayed round to oval nuclei , granular chromatin , occasional nucleoli , and moderate amount of granular to pale cytoplasm with areas of punctate necrosis and lympho - vascular emboli suggestive of thymic ne carcinoma . immuno - histochemical studies were suggestive of positive staining for chromogranin and synaptophysin , but absence of staining for nse and vimentin [ figure 4c and d ] , consistent with thymic ne carcinoma . lobulated solid tumor which was homogenous grayish white and fleshy on the cut surface histopathology and immuno - histochemistry . ( a ) tumor with adjacent thymus , h and e , 100 . ( b ) tumor cells displaying rosette formation and necrosis , h and e , 400 . ( c ) ( d ) cytokeratin immuno - positivity 200 with strong suspicion of sporadic men-1 , in view of coexistence of phpt with metastatic thymic ne carcinoma , work up for men-1 was done , which revealed raised fasting serum gastrin ( 18,000 pg / ml , normal < 200 ) suggestive of gastrinoma . his serum prolactin ( 12 ng / ml , normal 2.1 - 17.7 ng / ml ) and serum igf-1 ( 131 ng / ml ) were within normal range , thereby ruling out any functioning pituitary tumors . there was no family history of disorders suggestive of men-1 ( family pedigree chart , figure 5 ) . somatostatin - receptor scintigraphy with ga - dotanoc pet / ct revealed somatostatin receptor expressing tumors involving second part of duodenum , head , body , and tail of pancreas , suggestive of gastrinomas . there was no evidence of somatostatin receptor expression in the mediastinum or vertebrae , suggesting that the vertebral metastasis were from the thymic ne carcinoma and not from the gastrinomas . family pedigree chart showing index case of men-1 and unaffected family members the diagnosis of sporadic men-1 , with two major endocrine gland involvement ( phpt due to parathyroid hyperplasia and gastrinomas ) along with a rare occurrence of thymic ne carcinoma was apparent . the thymic ne carcinoma had evolved rapidly within a span of 8 months after parathyroidectomy and tct , and presented with bony metastases . with wide - spread metastatic disease , the patient was managed palliatively ( pantoprazole , zoledronic acid 4 mg intravenous infusion 4 weekly and lanreotide 20 mg intramuscular 4 weekly ) . the patient has been followed up with serum alkaline phosphatase , calcium , chromogranin and gastrin estimations ; and ga - dotanoc pet / ct and tc mdp bone scans at 6 monthly intervals , and other appropriate imaging such as abdomnal and thoracic ce - ct scans . he has been treated with external beam radiotherapy as well as sm samarium therapy for palliation of painful spinal lesions . sunitinib maleate- a multi - target tyrosine kinase inhibitor- 37.5 mg per orally , once daily was initiated in view of progressive metastatic disease , including liver , lungs and skeletal metastases , which he has received for 18 months with effective symptom palliation , and manageable toxicity . he suffered osteo - necrosis of the jaw- a known complication of long - term zoledronic acid usage , which has been managed by discontinuation of inj zoledronic acid and other supportive care . four years since being operated upon for the thymic n - e carcinoma , the patient is alive , ambulatory , is reasonably symptom free with good quality of life , and is normocalcemic , with no loco - regional recurrence of the thymic tumor . thymic ne tumors have been variably reported in men-1 from 1% to 8% of cases . in recent studies , thymic ne carcinoma has emerged as a major cause of mortality in men-1 along with gastro - entero pancreatic tumors . thymic ne tumors in men-1 are commoner in males and smokers and are almost always hormonally inactive and diagnosed incidentally . they are malignant , aggressive tumors and are widely invasive and metastatic at presentation ( usually to bone ) . thymic ne tumors are never the presenting feature of men-1 and almost always occur after phpt , providing an opportunity for prophylaxis for these tumors with tct at the time of parathyroid surgery . while operating a phpt patient with multigland parathyroid disease , routine tct is usually performed to take care of the supernumerary parathyroid glands that can be found within the thymus gland in 15 - 20% patients . the utility of tct in preventing the thymic ne tumors in men-1 patients is a matter of debate . our patient developed thymic ne carcinoma despite tct being performed as part of his first operation ( sub - total parathyroidectomy ) . tct could not prevent thymic ne carcinoma in our patient , as also reported by others . this may be because tct results in removal of only 40 - 50% of thymic tissue . in our patient , the thymic ne tumor was arising from thymic limb low down in the left pulmonary hilar region , which could not have been removed by tct . more radical thymectomy procedures like the trans - sternal thymectomy or video - assisted thoracoscopic surgical thymectomy have not been reported as a prophylactic procedure in men-1 patients . usually , phpt is the first component of men-1 manifesting in the third to fourth decade . in contrast , our patient had a rapid presentation of thymic ne carcinoma , which was not evident on the chest radiograph done about 9 months earlier during management of phpt . the rapid pace of development of thymic ne carcinoma with bone metastases in our patient underscores its aggressiveness and metastatic potential . current guidelines for surveillance of men-1 recommend screening for thymic ne tumors once every 1 to 2 years with ct or mri of thorax , although currently annual screening is advised by newer studies . as men-1 patients undergo parathyroid surgery almost universally , tct is recommended for prevention of thymic ne tumors . a complete surgical excision through a trans - sternal route is the only curative treatment for thymic ne tumors . our patient did not have any direct invasion of adjacent structures inspite of its large size and the histological margins were reported uninfiltrated . in view of the multiple bone metastases , we report a sporadic men-1 patient cured of phpt , presenting with gastrinoma and aggressive metastatic thymic ne tumor that developed within a year after subtotal parathyroidectomy along with tct . men-1 patients need to be screened for thymic ne tumors by routine annual ct or mri of the thorax even after tct at the time of parathyroid surgery .

abstractthe national institutes of health 's ( nih ) commitment to improving health outcomes for women and men through rigorous science has been compromised by the lack of basic science evidence obtained from female animals . to correct this limitation , in june 2015 the nih announced expectations that sex , as a biological variable , be included into research design and analysis in studies of vertebrate animals and humans ( not - od-15 - 102 ) . scientists must take the responsibility to implement this directive . however , in doing so , there is a risk that attention could be restricted to only studies of direct comparison between female / women and male / men . by contrast , understanding how sex influences health and disease needs to take a programmatic approach that includes the study of sex - specific conditions . a programmatic approach will assure the advancement of knowledge to improve women 's health . thymic neuroendocrine ( ne ) tumors are a rare manifestation of multiple endocrine neoplasia syndrome type 1 ( men-1 ) . they are malignant and aggressive tumors and form a major cause of mortality in men-1 . transcervical thymectomy ( tct ) at the time of parathyroid surgery for primary hyperparathyroidism ( phpt ) in men-1 usually prevents thymic ne tumors . we report a 56-year - old nonsmoker male with sporadic men-1 who presented with thymic ne carcinoma developing rapidly within a span of 8 months after subtotal parathyroidectomy and tct for phpt . we present a brief review of literature on this rare ne malignancy , focusing on its occurrence despite tct . this case highlights the fact that thymic ne carcinoma may develop even after tct in men-1 . regular surveillance for these aggressive thymic ne tumors is mandatory even after tct in men-1 setting .